TESIS DE DOCTORADO Desarrollo de herramientas moleculares para su aplicación en la mejora de la trazabilidad de los alimentos Fátima C. Lago Soriano
2017
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Fátima C. Lago Soriano FátimaLago C. TESISDOCTORA DE la los trazabilidad alimentos de laaplicación mejora su de en 2017
Escuela Internacional de Doctorado
Fátima C. Lago Soriano
TESIS DE DOCTORADO
DESARROLLO DE HERRAMIENTAS MOLECULARES PARA SU APLICACIÓN EN LA MEJORA DE LA TRAZABILIDAD DE LOS ALIMENTOS
Dirigida por los Doctores:
Montserrat Espiñeira Fernández
Juan Manuel Vieites Baptista de Sousa
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AGRADECIMIENTOS
Cuando una etapa llega a su fin, es cuando por fin puedes mirar a atrás, respirar profundamente, y acordarte de aquellos que te acompañaron. Del mismo modo, es difícil entender los agradecimientos de una tesis hasta que pones el punto y final. Es en este momento cuando se puede percibir la gratitud que sientes a todas las personas que han estado presentes durante esa etapa, ya bien sea codo a codo o simplemente trayéndote un café calentito en el momento preciso. Pero también es cierto que, entre toda esa gente que ha estado ahí, hay pocas caras que se dibujan clara e intensamente en mi cabeza.
En primerísimo lugar, me gustaría dar las gracias de una manera muy especial a Montse por muchos, muchísimos motivos: por darme cariño y amistad desde el día en que nos conocimos; porque a lo largo de esta década hemos compartido muchísimos momentos alegres, acompañados de risas y carcajadas, pero también los más tristes de mi vida, inundados de lágrimas y angustia; por estar ahí para lo que sea, para todo, y tener siempre tendida su mano amiga; por escucharme una y otra vez, sin cansarse, y aconsejarme sabiamente; por confiar en mí y guiarme, no solo durante el desarrollo de esta tesis, sino también en mi formación y día a día; por su eterna paciencia;… y, sobre todo, por poner en mi vida al “morenocho”, ese pequeño loquito tímido que me comería a besos. Por todo ello, mi eterno agradecimiento.
Porque por él empezó todo, quisiera agradecer enormemente a Fran el darme la oportunidad de formar parte de su equipo. Y, ahora que nuestros caminos se han vuelto a cruzar, por transmitirnos paz y tranquilidad en nuestro día a día, por ser esa voz sabia que escuchas
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cuando estás perdido, por tus notas de humor en plena tempestad,… por ser como eres.
Sin vosotros, equipo, no hubiese sido posible la finalización de esta tesis. En resumidas cuentas, esta tesis es más vuestra que mia.
Quiero extender un sincero agradecimiento a mis directores de tesis, Juan Manuel Vieites Baptista de Sousa y Montserrat Espiñeira Fernández, así como a mi tutora, Paloma Morán Martínez, por su ayuda y sabios consejos, disponibilidad y colaboración.
No puedo olvidar a mis compañeras y amigas con las cuales he compartido horas de trabajo. Unas se han ido y otras han llegado. A todas, gracias por los buenos y malos momentos, y por aguantarme.
Mención especial para Deivi, mi amor, mi amigo, mi compañero de viaje en ésta y otras tantas aventuras, mi confidente, mi gran apoyo, mi “Choupanés de la Choupana”… mi vida. Mil gracias por hacerme siempre la vida mucho más fácil. A tu lado la tristeza se transforma en alegría y la soledad no existe. A mis soles, Mikel y Mauro, mis “Micifús”, porque a vuestro lado vuela el tiempo y lo sois todo para mí. Dais sentido pleno a mi vida.
A todos los que ocupan un lugar especial en mi vida, mis padres, mis hermanos, demás familia y amigos,… por vuestro apoyo, gracias por haber sabido disculpar mis ausencias o mis salidas de tono, y aun así siempre haber tenido una palabra de ánimo.
Gracias a todos!!!
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ÍNDICE
INTRODUCCIÓN ...... 11 IDENTIFICACIÓN DE ESPECIES ...... 17 MÉTODO FINS (Forensically Informative Nucleotide Sequencing) ...... 20 ETAPAS DE LA TÉCNICA FINS ...... 24 EXTRACCIÓN DEL ADN ...... 24 SELECCIÓN DEL MARCADOR MOLECULAR ...... 27 DISEÑO DE LOS CEBADORES ...... 29 AMPLIFICACIÓN ...... 32 SECUENCIACIÓN ...... 33 ANÁLISIS FILOGENÉTICO ...... 35 MATERIAL DE REFERENCIA ...... 37 BASES DE DATOS ...... 39 VALIDACIÓN DE LAS TÉCNICAS MOLECULARES...... 41 APLICACIONES DE LA TÉCNICA FINS ...... 43 VENTAJAS Y DESVENTAJAS DE LA TÉCNICA FINS ...... 44 OBJETIVOS ...... 47 Authentication of species in meat products by genetic techniques ...... 51 Genetic Identification of Horse Mackerel and Related Species in Seafood Products by Means of Forensically Informative Nucleotide Sequencing Methodology ...... 63 FINS methodology to identification of sardines and related species in canned products and detection of mixture by means of SNP analysis systems ...... 71 Development of a FINS- based method for the identification of skates species of commercial interest ...... 83 Authentication of the most important species of freshwater eels by means of FINS ...... 91
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Authentication of gadoids from highly processed products susceptible to include species mixtures by means of DNA sequencing methods...... 99 DISCUSIÓN ...... 113 EXTRACCIÓN DEL ADN ...... 116 SELECCIÓN DEL MARCADOR MOLECULAR ...... 118 DISEÑO DE CEBADORES ...... 121 ANÁLISIS FILOGENÉTICO ...... 122 VALIDACIÓN METODOLÓGICA ...... 123 APLICACIÓN A MUESTRAS COMERCIALES/ESTUDIO DE MERCADO...... 125 CONCLUSIONES ...... 131 REFERENCIAS ...... 139
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INTRODUCCIÓN
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INTRODUCCIÓN
En los últimos años, las técnicas de identificación de especies han adquirido gran relevancia en el sector de la alimentación. Este auge surge como respuesta al creciente interés de los consumidores por disponer de la mayor cantidad de información posible sobre los productos que se encuentran a su alcance en los mercados, demandando cada vez más, productos saludables, de calidad y con características nutricionales determinadas.
Tanto los productores y comercializadores, como las propias autoridades de control, conscientes de estos cambios, aplican estas técnicas en sus controles, incrementando la calidad y la seguridad alimentaria de dichos productos.
En este sentido, la trazabilidad se ha convertido en una prioridad en el sector alimentario. Es imprescindible para ofrecer transparencia y garantías a los consumidores, quienes exigen cada vez estándares más elevados de frescura de los alimentos, así como diversidad, conveniencia e inocuidad; y garantías de calidad como la rastreabilidad, las normas de empaquetado y los controles de elaboración.
Los consumidores piden garantías de que los alimentos hayan sido producidos, manipulados y comercializados de un modo que no sea perjudicial para su salud, que respete el medio ambiente y que aborde otras preocupaciones éticas y sociales [1]. Pero también es un aspecto fundamental para los productores, ya que les permite asegurar la calidad de la materia prima que introducen en la cadena alimentaria, así como la certificación de sus productos en base a distintos estándares, la rápida localización de partidas problemáticas y la
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implantación de sistemas control, evitando el fraude y la competencia desleal entre ellos.
En el Reglamento Europeo 652/2014 se define la trazabilidad como “la posibilidad de encontrar y seguir el rastro, a través de todas las etapas de producción, transformación y distribución de un alimento, un pienso, un animal destinado a la producción de alimentos o una sustancia destinada a ser incorporada en alimentos o piensos o con probabilidad de serlo” [2].
Un buen sistema de trazabilidad debe permitir seguir el rastro de un producto, tanto hacia atrás como hacia delante, a lo largo de toda la cadena alimentaria, sobre todo en el caso de que se produzca alguna incidencia que afecte a la calidad o la seguridad del producto. Las alertas alimentarias surgidas en los últimos años han creado una gran sensibilización social ante la necesidad de consumir alimentos seguros. Esto se ha traducido en una estricta legislación, que impone el control y la trazabilidad en las etapas de producción, transformación y distribución de alimentos. La importancia que el consumidor da a este hecho hace que el cumplimiento de estas normas vaya más allá de las imposiciones legales y se convierta en un elemento de calidad y marketing que aporta valor diferencial a los productos.
Los sistemas de gestión de la calidad permiten conformar un régimen de controles y registros que funcionan como detectores de incidencias, abarcando toda la cadena de producción. El control de proveedores, materias primas y competidores, así como la trazabilidad del alimento, ofrece ventajas mucho más allá: permite obtener los estándares de calidad requeridos por los clientes, diferenciar los productos e imponer la marca, mejorar la posición en los mercados más exigentes, dar mayor valor añadido a los productos, afianzar la posición comercial y facilitar
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el acceso a nuevos mercados, redes de distribución, consumidores, etc. que buscan comprar con confianza [3].
Además, las normativas relacionadas con la rotulación y etiquetado de los alimentos han ido apareciendo y desarrollándose cada vez de forma más exigente. En este sentido, con el fin de proteger a los consumidores y evitar el fraude en el mercado, la Unión Europea ha establecido el Reglamento Europeo (CE) 1379/2013 [4] donde se regula el correcto etiquetado de los alimentos. En este reglamento se otorga un carácter prioritario al hecho de que el consumidor posea una adecuada información sobre el producto que va a consumir.
Las disposiciones de aplicación de este reglamento son desarrolladas posteriormente en el Reglamento de Ejecución (UE) nº 1420/2013 [5], donde se detallan minuciosamente las normas de etiquetado y la información mínima necesaria que debe ser incluida para conocimiento del consumidor, tales como la denominación comercial y el nombre científico de la especie, así como el método de producción y la zona de captura u origen. Así, el cumplimiento de estas normas permite la elección a los consumidores y proporciona información completa y precisa sobre el producto a adquirir.
De este modo, tanto el cumplimiento de estas normativas como la implantación de los sistemas de trazabilidad a lo largo de toda la cadena alimentaria, permiten disponer de una información completa y veraz de los productos comercializados, y este hecho confiere una mayor transparencia del mercado, lo cual ayuda a alcanzar una mayor estabilidad a fin de asegurar el control de la calidad comercial e incrementar la transparencia a lo largo de la cadena alimentaria, facilitando la apertura de mercados, la internacionalización de multitud de productos y el crecimiento global de la industria alimentaria, además
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de evitar tanto el fraude al consumidor como la competencia desleal entre productores [3].
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IDENTIFICACIÓN DE ESPECIES
Durante años, la identificación de especies ha estado fuertemente ligada a la identificación en base a caracteres morfológicos mediante el uso de claves taxonómicas, que posibilitaba el reconocimiento y diferenciación de especies. Pero, a pesar de ser una técnica que no requiere equipos sofisticados ni la alteración de la materia prima, manteniéndola intacta y en buenas condiciones para su venta y consumo, este método depende de la habilidad y la cualificación del personal técnico. Además, cuando los caracteres morfológicos de la materia prima han cambiado completamente debido a las diferentes etapas de procesamiento, esta identificación se hace difícil, y en la mayoría de los casos, imposible.
Con el fin de salvar estos inconvenientes, surgen las técnicas moleculares, que se basan en componentes intrínsecos de los tejidos y son independientes de caracteres morfológicos. Las técnicas moleculares pueden ser divididas principalmente en dos grupos: técnicas basadas en proteínas y técnicas basadas en ácidos nucleicos.
Los métodos analíticos convencionales más utilizados para el análisis de alimentos basados en proteínas incluyen métodos como los electroforéticos, cromatográficos, espectroscópicos e inmunológicos. Sin embargo, la principal limitación de estas técnicas se presenta cuando los productos han sido sometidos a tratamientos térmicos que desnaturalizan las proteínas, imposibilitando su aplicación.
Las técnicas basadas en el análisis de ácidos nucleicos y, concretamente, las basadas en la Reacción en Cadena de la Polimerasa (PCR), presentan importantes ventajas frente a las basadas en el análisis de proteínas. Entre ellas destacan: la pequeña cantidad
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de muestra requerida para el análisis; el mayor grado de variabilidad, es decir, los distintos cambios que se pueden producir en el material genético dentro de una misma especie y entre especies; y la posibilidad de analizar muestras sometidas a diferentes tratamientos de procesado, debido a la elevada estabilidad del ADN.
El proceso de identificación de especies mediante las técnicas genéticas se basa en la identificación de secuencias de ADN específicas para cada una de las especies. Esto permite determinar con fiabilidad y rapidez las materias primas incluidas en los alimentos y productos alimenticios.
El ADN ofrece una serie de ventajas cuando se compara con las proteínas. En primer lugar, el ADN que contienen todas las células de un individuo es idéntico, independientemente del tejido. En cambio, las proteínas, son el resultado de la expresión génica, que puede sufrir variaciones de unos tejidos a otros, de una etapa de desarrollo a otra, de un medio ambiente a otro, y de una época del año a otra. Además, la naturaleza degenerada del código genético hace que existan más tripletes o codones que aminoácidos, de forma que un determinado aminoácido puede estar codificado por más de un triplete, lo que implica la existencia de mayor variabilidad del ADN que de las proteínas, siendo por tanto, más informativo.
El ADN es una molécula termoestable y, aunque se degrada durante el proceso de transformación (fragmentación del ADN), los fragmentos generados siguen siendo informativos. Es importante destacar que en el proceso de elaboración de las conservas, se emplean temperaturas y presiones elevadas, por ello se producen fragmentos de entre 100 y 250 pares de bases (pb) aproximadamente [6]. Las proteínas, en cambio,
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sufren desnaturalización, impidiendo su utilización, por lo que ya no son útiles para las técnicas de identificación de especies.
Es importante tener en cuenta una serie de factores en la identificación de especies. Las regiones de ADN seleccionadas deben acumular mutaciones a una velocidad suficiente como para que especies estrechamente relacionadas presenten secuencias de nucleótidos diferentes, permitiendo su diferenciación; pero al mismo tiempo, la velocidad tiene que ser lo suficientemente lenta como para que las diferencias no aparezcan dentro de la misma especie. La longitud del fragmento amplificado es otro aspecto clave. Éste ha de ser lo suficientemente grande como para detectar diferencias interespecíficas. El nivel de polimorfismo de los marcadores genéticos condicionará su posible aplicación para el estudio de relaciones entre especies, poblaciones o individuos [3].
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MÉTODO FINS (Forensically Informative Nucleotide Sequencing)
Bartlett y Davidson propusieron la identificación genética de especies mediante el análisis filogenético de secuencias de ADN en 1992. La denominación de esta técnica recibe su nombre de las siglas en inglés “Forensically Informative Nucleotide Sequencing”, es decir, Secuenciación de Nucleótidos con Información Forense, con lo que estos autores resaltan el componente forense de esta metodología [7]. Este método se divide en varias etapas: extracción de ADN, amplificación por PCR de un fragmento de ADN y secuenciación.
La Ciencia Forense es la aplicación del conocimiento de la ciencia, la ingeniería y el arte en la resolución de aspectos relacionados con la ley [8]. Según la Forensic Science Society del Reino Unido, la Ciencia Forense es la aplicación de los conocimientos científicos a cuestiones legales, entendiendo como cuestiones legales todo suceso que pueda requerir la intervención de un científico y que, dependiendo de la decisión de un juez o un jurado, haya que investigar las causas que excedan de los conocimientos que aporta el derecho (un robo, una falsificación de documentos, el derrumbamiento de un edificio, un vertido a un río, un homicidio,...).
Actualmente, la Ciencia Forense comprende una amplia diversidad de disciplinas científicas que trabajan de manera especializada e interdisciplinar para el cumplimiento de un objetivo común, responder a preguntas de interés legal en relación con un delito o una acción civil, mediante la evaluación de las evidencias.
Una de estas disciplinas es la Biología Forense. Esta especialidad se encarga de la identificación y comparación de seres vivos mediante el estudio de fluidos, tejidos y estructuras biológicas. La Biología Forense
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incluye, entre otras áreas, la identificación de individuos, la determinación de la paternidad, la identificación de especies animales y vegetales,… El biólogo forense es el encargado de analizar, identificar e individualizar vestigios biológicos para poder así colaborar en la aclaración de determinados hechos.
Dentro de la Biología Forense, la Genética Forense emplea técnicas moleculares basadas en el análisis del ADN para la identificación de individuos. Además, hay que destacar que este análisis no será veraz si no es posible demostrar que las muestras se manejan de acuerdo a un protocolo aprobado. Es necesario que existan determinados controles que eviten contaminaciones de la muestra. Como en cualquier Ciencia Forense, el manejo de las evidencias físicas es uno de los factores más importantes de la investigación y, por ello, siempre se siguen unos protocolos de recogida, transporte, inventariado, conservación y análisis, con el fin de proteger la cadena de custodia de muestras.
En España, desde sus inicios a principios de los años 90, esta disciplina no ha dejado de evolucionar, y sigue en constante desarrollo. Las técnicas basadas en el estudio del ADN se han convertido en una de las herramientas más precisas, revolucionado la investigación policial y los sistemas de identificación. Gracias a estos avances, hoy en día se pueden resolver casos que hace algunos años no serían abordables.
En la alimentación actual destacan los productos elaborados, sobre todo aquellos que se adecuan a nuestro ritmo de vida. Además, se busca mantener una alimentación sana y equilibrada, acentuando el consumo de alimentos ricos en proteínas, tales como las carnes y los pescados. Por ello, la identificación de especies en alimentos, se ha convertido en un tema muy necesario dentro de la industria alimentaria y entre los consumidores, entre otras razones, para evitar la sustitución o
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adulteración de especies dentro de un producto; o por motivos de salud humana, tales como alergias alimentarías; también se puede relacionar con riesgos para la subsistencia de especies protegidas.
Los argumentos anteriores enfatizan la necesidad de desarrollar técnicas que permitan verificar la especie a partir de la que se ha elaborado un producto. Así, las técnicas forenses aplicadas a la identificación de especies, hacen referencia a las herramientas a través de las cuales podemos llegar a la identificación de una muestra desconocida siguiendo su huella genética.
La huella genética es una técnica utilizada para distinguir entre individuos utilizando su ADN. Esto es posible ya que existen posiciones variables en el ADN de diferentes especies/individuos, conocidos como polimorfismos, y que constituyen el perfil genético de cada especie/individuo, permitiendo su identificación inequívoca.
Bartlett y Davidson fueron pioneros en la aplicación de la PCR y posterior secuenciación de DNA de muestras desconocidas, para luego compararlas con secuencias de especies conocidas, con el fin de resolver la identidad de la especie problema.
La base de esta técnica es la comparación de secuencias de muestras desconocidas con secuencias de referencia o patrón. A partir del conjunto de secuencias de la especie desconocida y secuencias de referencia. Se realiza el análisis filogenético y los resultados se pueden visualizar a través de una filogenia o árbol filogenético. Este árbol se construye usando diferentes métodos y agrupa las secuencias más similares próximas, y más alejadas a medida que aumentan las diferencias, permitiendo asociar la especie desconocida según el nodo en el que se posicione. Entre los métodos de construcción de árboles más usados con esta finalidad destaca el de Neibourg-Joining [9]. La
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fiabilidad de las agrupaciones se evalúa mediante la aplicación del test de Bootstrap. Este método se usa para obtener un soporte estadístico de los diferentes grupos obtenidos en el árbol filogenético.
El estudio de Bartlett y Davidson fue muy novedoso ya que habían desarrollado con éxito una de las primeras metodologías de diagnóstico para identificación de numerosas especies, no solo de pescado sino también de diferentes mamíferos y aves, basándose en la secuenciación de ADN, que era considerada la forma más directa de obtener una gran cantidad de información.
Por aquel entonces, el desarrollo de este tipo de metodologías no era fácilmente accesible debido al coste de la tecnología empleada. Además, el conjunto de secuencias de referencia para la comparación era muy limitado. Hoy en día, casi tres décadas después, los grandes avances tecnológicos y el estudio de diferentes grupos taxonómicos hacen posible la aplicación de esta tecnología en cualquier laboratorio medianamente equipado, permitiendo obtener resultados de identificación muy precisos.
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ETAPAS DE LA TÉCNICA FINS
EXTRACCIÓN DEL ADN
Proceso mediante el cual se obtiene esta biomolécula tras aislarla del resto de componentes celulares.
El aislamiento y purificación del ADN constituye un paso crucial debido a que, si la extracción no se realiza correctamente (si la cantidad y calidad de los ácidos nucleicos obtenidos no es suficiente), es posible que el resto de etapas no tengan éxito.
Existen diferentes métodos que permiten el aislamiento y purificación del ADN, entre ellos cabe destacar [11]:
Extracción con solventes orgánicos: Método convencional de amplio uso donde, tras la lisis de las células, el resto de componentes celulares generalmente son eliminados mediante sucesivas precipitaciones con solventes orgánicos (tales como fenol, cloroformo, isopropanol o etanol) y centrifugaciones. El ADN purificado suele ser recuperado mediante precipitación con etanol ya que en presencia de cationes monovalentes como los de Na y a temperatura de -20°C, precipita eficientemente dejando atrás ácidos nucleicos monoméricos y de cadena corta.
Extracción mediante columnas de sílica: Método ampliamente utilizado en los kits comerciales actuales. El ADN se adsorbe específicamente a membranas/esferas/partículas de sílica en presencia de ciertas sales y a un pH particular, mientras que los restos celulares son removidos por sucesivos pasos de lavado. El ADN es finalmente eluído
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en un tampón de baja salinidad. Su procedimiento es más simple y rápido que la extracción orgánica y además puede ser automatizable.
Extracción magnética: Método basado en la unión reversible del ADN a partículas sólidas magnéticas recubiertas de sílice. Tras esta unión, los elementos magnéticos son separados del resto de componentes celulares mediante sucesivos lavados y finalmente el ADN purificado es eluído. Este método es rápido y ofrece la posibilidad de ser automatizado.
En la actualidad, existen multitud de kits comerciales que incorporan en sus protocolos tanto los métodos de extracción basados en columnas de sílica como en partículas magnéticas, y que permiten una agilización, estandarización y automatización del procedimiento de aislamiento del ADN.
Una vez extraído el ADN se ha de medir la cantidad y calidad del ADN obtenido. La aproximación más usual es la espectrofotométrica mediante la medición de la absorbancia a 260 nm, lo que permite obtener la concentración de la solución de ADN obtenida ya que el ADN presenta su máximo de absorción a esta longitud de onda. La ratio de absorbancias a 260 y 280 nm (A260/A280) indica la proporción de ácidos nucleicos en relación a proteínas (las proteínas tienen su máximo de absorción a 280 nm). Estos valores deben estar comprendidos entre 1,7 y 2,0, indicando que hay el doble de ácidos nucleicos en relación a proteínas en la extracción. Valores inferiores pueden indicar la presencia de impurezas o contaminantes en la solución como resultado de determinados métodos de extracción, como pueden ser presencia de proteínas o de restos de fenol, y que pueden afectar al ensayo. Valores superiores pueden ser debidos a la presencia de ARN [12].
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El grado de fragmentación del ADN puede ser estimado mediante la separación electroforética en geles de agarosa, donde el ADN de mayor peso molecular aparece como una banda compacta próxima al pocillo de carga, mientras que el ADN degradado presenta un barrido de bandas a lo largo de la calle del gel.
Es importante destacar que, a baja temperatura, el ADN tiende a ser muy estable, sin embargo, se fragmenta durante los procesos de elaboración de diferentes productos, tales como la esterilización, donde se aplican elevadas temperaturas sumadas a la sobrepresión. Con estos tratamientos, el ADN se degrada en fragmentos de en torno a los 200 pb [6].
Por otro lado, la presencia de ciertas especias, salsas, ácidos, grasas y otros aditivos producen una disminución del pH que también puede derivar en degradación del ADN, además de favorecer la presencia de sustancias inhibidoras de la PCR en la extracción, reduciendo la eficiencia de la amplificación [13].
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SELECCIÓN DEL MARCADOR MOLECULAR
Los marcadores moleculares son segmentos de ADN situados en lugares concretos del genoma, empleados para “marcar” y poder rastrear una posición concreta del genoma. Estos marcadores pueden ser genes o simplemente una secuencia de nucleótidos sin función conocida.
La correcta selección del marcador molecular para la amplificación por PCR es otro de los factores cruciales en los métodos FINS, debido a que la resolución de esta técnica depende en gran medida de la variabilidad y el número de sitios informativos de la secuencia seleccionada.
La tasa de evolución de un marcador molecular es la cantidad de cambios que sufre en relación al tiempo. Estas tasas varían entre las diferentes regiones del ADN, pudiendo obtener resultados con mayor resolución al emplear regiones con suficiente variabilidad. De esta forma, regiones que tienen una alta tasa de evolución pueden ser útiles en la identificación a nivel de especie, mientras que regiones con una tasa baja solamente permitirán identificar a nivel de género o de familia. La tasa de evolución de una misma región del ADN puede variar entre organismos, es decir, puede ser ideal para la identificación específica de especies animales sin embargo, no ser adecuado para la mayoría de plantas, como es el caso del COI (Citocromo Oxidasa subunidad 1) ya que en éstas, posee pocos sitios polimórficos, probablemente debido a una tasa de mutación baja [14].
Cuando las muestras proceden de productos que han sido sometidos a procesos de transformación, la selección del marcador molecular se ve dirigida fundamentalmente a genes mitocondriales. Esto es debido a que son multicopia, es decir, tiene mayor abundancia en la célula que
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el ADN nuclear. Además, algunos autores han sugerido que, el ADN mitocondrial, al ser circular, tiene una mayor resistencia intrínseca frente a la fragmentación por calor [15].
Una opción común en el desarrollo de metodologías mediante la técnica FINS es el uso del marcador mitocondrial Citocromo b (Cytb) y COI. El primero ha sido empleado con éxito en la identificación de diversos grupos taxonómicos [6, 13, 16-22], al igual que el segundo, [23, 24]. Además, estos marcadores son utilizados por varias plataformas centradas en la identificación de especies como son FISHTRACE (Cytb) y BARCODE OF LIFE (COI) [25, 26].
Otros marcadores que también se han empleado son: el gen mitocondrial 16S ARNr [27-29]; el gen mitocondrial 18S ARNr [22, 30-32]; las regiones del espaciador interno transcrito (ITS 1 y 2) [33-35]; o el gen nuclear 28S ARNr [36].
En resumen, un buen marcador molecular debe tener una alta variación interespecífica, a la vez que una baja variación intraespecífica. Debe tener suficientes sitios polimórficos para que permita alinear la secuencia desconocida con las secuencias de referencia y poder así obtener una identificación precisa e inequívoca.
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DISEÑO DE LOS CEBADORES
Existen numerosos paquetes informáticos disponibles para el diseño de cebadores para la PCR. Algunos ejemplos son: AlleleID [37], Beacon Designer (Premier Biosoft), o Primer Express (Applied Biosystems). Todos ellos se basan en algoritmos que aplican una serie de reglas para el diseño. En general son de fácil uso, inicialmente se introducen las secuencias donde se quieren diseñar los cebadores y el programa permite realizar un diseño dirigido o automático. Además, recomiendan unas concentraciones de reactivos en la PCR, así como unos ciclos térmicos, minimizando la puesta a punto y optimización de la PCR.
Una serie de recomendaciones para el diseño de los cebadores serían los siguientes:
Su longitud óptima oscila en general entre los 15-25 pb. Para maximizar la especificidad, el contenido óptimo de G/C debería estar en torno al 50% pero el rango puede ampliarse entre 30- 80%. Secuencias con un alto contenido en G/C puede reducir la eficiencia y producir productos no específicos [38]. La temperatura media de los cebadores debería estar entre 58 y 60ºC y la diferencia entre el cebador directo y el reverso no debería ser mayor de 1-2ºC. El riesgo de uniones inespecíficas puede ser reducido mediante el diseño de cebadores que solo contengan entre 1-3 G/C dentro de las últimas 5 bases en el extremo 3’ [38, 39]. Los cebadores no deben contener 6 o más nucleótidos iguales contiguos, ni contener ningún nucleótido ambiguo, y debe evitarse la autocomplementariedad [40].
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Una vez diseñado el cebador, es necesario seleccionar el método de purificación para el proceso de síntesis, donde, en cada adición de base aproximadamente el 1% de los oligonucleótidos no incorporan la base adecuada. Esto da lugar a una mezcla de cebadores de longitud total y productos truncados (de menor longitud) [41]. Estas secuencias incompletas pueden competir con el cebador completo en la PCR por lo que es necesario purificarlos. Existen diversos métodos de purificación y su elección dependerá de la aplicación a la que destinados los cebadores [42]:
Desalado: los oligonucleótidos son tratados con una columna de cromatografía que remueve las sales pero no las secuencias erroneas. Proporciona una solución de ADN libre de sales, indicada para PCR y aplicaciones de secuenciación sin posterior purificación ya que los truncamientos y supresiones no afectan al resultado.
Cromatografía de fase reversa: las secuencias falladas son eliminadas. Esta purificación está indicada para oligonucleótidos de gran tamaño sin modificaciones, ya que los fragmentos más cortos y las sales son eliminadas.
HPLC (Cromatografía líquida de alto rendimiento): Remueve las secuencias erróneas o marcaciones no incorporadas. Garantiza oligonucleótidos altamente purificados (>85% completos). Indicado para oligonucleótidos modificados y no modificados de hasta 60 bases.
PAGE: Método usado para diferenciar secuencias completas de las incompletas, basado en el tamaño y la conformación. Provee el porcentaje más alto de oligonucleótidos completos (>85%) requerido para ciertas aplicaciones como mutagénesis o clonación. Sin embargo,
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puede dañar algunas modificaciones, incluyendo fluoróforos y algunas modificaciones utilizadas para la ligación.
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AMPLIFICACIÓN
Casi todas las tecnologías basadas en el estudio del ADN para la autentificación de diferentes especies se basan, al menos en una primera etapa, en la amplificación de una región concreta de ADN mediante PCR.
La PCR o Reacción en Cadena de la Polimerasa fue desarrollada en 1986 por Kary Mullis. Su objetivo es obtener un gran número de copias de un fragmento de ADN particular, partiendo de una única copia de ese fragmento original o molde [43].
La PCR se puede dividir en 3 fases: desnaturalización, hibridación de los cebadores sobre el molde y extensión de los cebadores. Para que se pueda copiar cada hebra del ADN (inicialmente en forma de doble hélice) es necesario que se rompan los enlaces existentes entre estas dos hebras. Este proceso se realiza elevando la temperatura aproximadamente a 95ºC durante un breve período de tiempo (desnaturalización). A continuación, se necesita que los cebadores se unan a sus secuencias complementarias. En este paso la temperatura juega un papel importantísimo, puesto que cada pareja de cebadores hibrida a una temperatura que, generalmente, puede variar entre 45 y 65ºC (hibridación). Finalmente, se necesita la extensión de los cebadores gracias a la acción de la enzima (polimerasa) y que generalmente tiene su temperatura óptima de reacción a 72ºC (extensión). En cada ciclo de amplificación, el fragmento de ADN diana aumenta su número de copias de forma exponencial. De este modo, al final de un programa básico de PCR, se obtienen hasta 100 millones de copias del fragmento deseado.
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SECUENCIACIÓN
La secuenciación del ADN es el método que permite obtener la mayor cantidad de información de las regiones amplificadas por PCR, y la secuencia completa de los nucleótidos caracterizados por sus bases nitrogenadas: Adenina (A), Timina (T), Citosina (C) y Guanina (G).
Hasta hace poco, el método más usado para la secuenciación de ácidos nucleicos era el método enzimático de terminación de cadena de Sanger o método didesoxi, diseñado por Fred Sanger en 1977, aunque actualmente se están extendiendo los equipos de secuenciación masiva basados en distintas tecnologías, según la empresa que los desarrolla. Generalmente, esta tecnología se basa en la inmovilización del ADN molde en un sistema de soporte sólido para, posteriormente, amplificar esos fragmentos mediante PCR y secuenciarlos de forma paralela y masiva. El resultado es una tecnología de secuenciación que permite la secuenciación de genomas enteros [44].
En el método de Sanger se utilizan los dideoxinucleótidos (ddNTP), que se diferencian de los deoxinucleótidos (dNTP) en que carecen del grupo OH en el carbono 3’ mediante el que se extiende la hebra de ADN por las polimerasas. Por ello, cuando se unen ddNTP, no permite la incorporación de un nuevo nucleótido, y la replicación se paraliza.
En la secuenciación se parte de ADN bicatenario amplificado por PCR, al que se añade: Taq polimerasa, los cuatro tipos de deoxinucleótidos (A, G, T, C); un cebador que actúa como sustrato de la ADN polimerasa; y una pequeña cantidad de cada uno de los ddNTP marcados cada uno con un fluoróforo, que se incorporarán al azar en competición con su dNTP correspondiente.
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Estos fluoróforos van a excitarse con luz, emitiendo luz de una longitud de onda característica que es detectada por los secuenciadores automáticos, permitiendo asignar cada base de la secuencia de ADN.
Los secuenciadores funcionan de forma completamente automática, inyectando las muestras en un capilar previamente cargado con un polímero, que funciona como lo hace el gel desnaturalizante de acrilamida. A una altura determinada del capilar, el láser detecta la fluorescencia emitida por cada cadena sencilla de ADN fluorescente y traduce esta emisión de fluorescencia en la secuencia correspondiente. Una vez desarrollada la electroforesis de la primera muestra, el capilar se vacía, se rellena nuevamente con polímero e inyecta una nueva muestra, y así sucesivamente.
Estos equipos permiten la automatización completa de este proceso, leyendo del orden de 1 kb en el plazo de una hora por cada capilar.
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ANÁLISIS FILOGENÉTICO
Los métodos FINS se basan en la comparación de secuencias de ADN de muestras desconocidas frente a secuencias de especies de referencia.
Las secuencias son obtenidas a partir de especímenes patrón perfectamente caracterizados, cedidos por científicos de diferentes instituciones, universidades, museos y centros tecnológicos distribuidos por todo el mundo. Además, existen distintas bases de datos de libre acceso como es el caso de la base de datos del NCBI (GenBank) de las que es posible obtener secuencias [45]. Sin embargo, la calidad y veracidad de éstas no siempre está garantizada [46, 47].
El análisis filogenético se realiza incluyendo tanto las secuencias de las muestras desconocidas como las de referencia.
Este análisis puede realizarse por diferentes métodos, entre los que se pueden destacar:
Métodos de distancias, de los cuales Neighbor Joining (NJ) es el más utilizado, aunque hay otros (UPGMA y Mínima evolución). Máxima parsimonia. Máxima probabilidad. Bayesiano.
De entre todos ellos, el primero es el más utilizado.
En estos métodos de distancias es posible seleccionar el modelo de sustitución nucleotídica, desde modelos simples a más complejos, que incluyen más parámetros, ajustándose mejor a los datos observados. Como resultado del análisis se obtiene una matriz de distancias, que muestra la distancia genética entre pares de secuencias. A partir de
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esta matriz, se construyen las filogenias o árboles filogenéticos, en los que se muestra la similitud de secuencias de forma gráfica, ya que se agrupan las muestras pertenecientes a especies próximas en un mismo nodo o clado. De este modo, se asignan las muestras desconocidas a un determinado grupo taxonómico, según su posición en dicho árbol.
Para comprobar la fiabilidad de las agrupaciones generadas, se emplea el test de Bootstrap. Éste es un método de remuestreo con reemplazamiento que construye diferentes alineamientos al azar, reemplazando un número determinado de posiciones nucleotídicas con otras posiciones del alineamiento, cada una de las cuales tiene la misma probabilidad de reemplazar a las demás. Por tanto, en el nuevo alineamiento, un sitio puede estar repetido más de una vez a costa de otros sitios. A partir de cada uno de los nuevos alineamientos se infiere un nuevo árbol filogenético, utilizando el mismo método empleado que en el alineamiento inicial. El porcentaje de veces que cada rama interior del árbol inicial se confirma en el conjunto de los árboles obtenidos por bootstrap, constituye el valor de bootstrap de cada rama. Valores mayores o iguales a 70 se corresponden con una probabilidad igual o mayor al 95% de que el clado o nodo sea real [10].
De esta manera, se comprueba la fiabilidad de cada una de estas agrupaciones y, por tanto, la robustez de los métodos.
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MATERIAL DE REFERENCIA
Una de las tareas más laboriosas e importantes, en el desarrollo de estas metodologías de identificación de especies es la obtención de Materiales de Referencia (MR).
En ausencia de Material de Referencia Certificado (MRC), y dada la escasez de MR comerciales para los procedimientos de identificación de especies, es necesaria la preparación, caracterización y determinación de Materiales de Referencia Internos (MRI), que son aquellos preparados por el laboratorio para su propio uso. Éste debe poder garantizar la fiabilidad y la precisión de las determinaciones realizadas, por lo que deben ser caracterizados exhaustivamente para poder convertirlos en MRC trazable.
Por otro lado, todos estos MR se engloban en Bancos de Tejidos (BT) con el objetivo de almacenar los materiales biológicos a partir de los que se pueda extraer ADN en cantidad y calidad suficiente, de modo que pueda ser empleado en los análisis de identificación de especies mediante técnicas moleculares. Los BT se componen de dos partes: una que almacena físicamente el conjunto de muestras, y que sirven como MR para la determinación de otros especímenes; y la otra archiva electrónicamente o en otros soportes toda la información de dichos MR.
Los MR pueden ser conservados con diferentes métodos: ultracongelación (<-40ºC), donde se conservan los tejidos, ejemplares completos (cuando su tamaño lo permite); o la propia extracción de ADN; liofilización de tejidos y posterior ultracongelación; y en fluido, donde se conservan fracciones de tejidos principalmente en etanol absoluto con posterior ultracongelación. La cantidad de tejido necesaria para realizar una extracción de ADN es pequeña (un
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microtubo puede ser utilizada para multitud de determinaciones y, de cada uno de ellos, se obtiene gran cantidad de ADN, con lo que prácticamente es un recurso inagotable).
Las secuencias obtenidas a partir del análisis de distintos marcadores moleculares de estos MR se almacenan en bases de datos de secuencias. Además, también sirven para estudios taxonómicos, permitiendo la identificación y clasificación de numerosas especies.
Los BT son un archivo histórico natural de utilidad múltiple, donde la preservación de las muestras y su información asociada son la base de la genética de poblaciones, taxonomía, sistemática, ecología, estudios filogenéticos, biogeográficos y de conservación. Todos ellos son una parte fundamental del conocimiento de la diversidad biológica.
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BASES DE DATOS
Una Base de Datos es un conjunto de registros pertenecientes a un mismo contexto. En este sentido, el éxito de la identificación de especies mediante FINS depende, en gran medida, de las secuencias patrón obtenidas a partir de las especies de referencia.
Existen diferentes programas denominados Sistemas Gestores de Bases de Datos que permiten almacenar y posteriormente acceder a los datos de forma ordenada y estructurada. BioloMICS es uno de estos sistemas, permite además del almacenamiento y gestión de datos, su análisis.
Este software permite la creación y mantenimiento de bases de datos, organizando la información obtenida a partir de todos los especímenes patrón. Además, BioloMICS permite comparar registros desconocidos con un número ilimitado de registros de referencia en base a cualquier campo almacenado, es decir, en base a características morfológicas, fisiológicas, datos moleculares, químicos, ecológicos, geográficos,…
Además, posee una serie de funciones relacionadas con la Biología Molecular entre las que destacan:
Almacenamiento de secuencias de ADN para su posterior edición y análisis: permite crear una base de datos de secuencias. Alineamientos múltiples de secuencias para realizar identificaciones y clasificaciones: permite alinear secuencias desconocidas en base a las secuencias almacenadas en la base de datos local y, al mismo tiempo, con las secuencias depositadas en la base de datos del NCBI. Estos alineamientos de secuencias se pueden almacenar, editar, exportar...
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Creación de árboles filogenéticos basados en distintos métodos tales como: Neighbor Joining, UPGMA, UPGMC, WPGMA…etc. Almacenamiento, edición y análisis de geles de electroforesis, así como el cálculo de pesos moleculares y distancias de migración.
La principal desventaja de BIOLOMICS es el coste, por ello hay organismos que proporcionan Bases de Datos online gratuitas. Entre estas destaca la del National Center for Biotechnology Information (NCBI). Este organismo dispone de una página web muy útil compuesta por múltiples bases de datos interconectadas (de secuencias, de proteínas, de genomas, taxonómica, de publicaciones,…), de tal forma que se puede usar un único sistema de búsqueda y recuperación de datos llamado ENTREZ [48]. Este sistema de búsqueda permite que un usuario tenga acceso y recupere información de muchas bases de datos a la vez.
Otro de los recursos que ofrece el NCBI es la herramienta BLAST (Basical Logical Alignment Search Tool), que permite la posibilidad de comparar una secuencia determinada con las secuencias presentes en las bases de datos de manera automática, permitiendo asociar una secuencia desconocida con un determinado grupo taxonómico [49]. Para ello, se introduce la secuencia desconocida en la ventana de búsqueda de esta herramienta y se obtiene una lista de las secuencias almacenadas con mayor homología a la secuencia introducida, ordenada de mayor a menor grado de similitud. De ahí la importancia de poseer Bases de Datos lo más amplias y completas para que la identificación sea posible.
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VALIDACIÓN DE LAS TÉCNICAS MOLECULARES
Una parte fundamental en cualquier desarrollo de una herramienta molecular que va a ser aplicada a productos comerciales procesados es la validación. El principal objetivo de esta etapa del desarrollo metodológico es verificar que ningún factor al que se ven sometidos los productos durante el proceso de transformación pueda influir en la aplicación de los métodos desarrollados. Para ello, es necesario elaborar distintos productos a partir de individuos pertenecientes a especies autentificadas en base a caracteres morfológicos, genéticas,…
De esta forma, se preparan los productos elaborados, aplicándoles los principales tratamientos que se utilizan en la industria alimentaria. Dichos tratamientos incluyen congelación, esterilización, ahumado, marinado, carpaccio, y platos precocinados, con diferentes salsas y condimentos.
El tratamiento más agresivo es el aplicado a las conservas, que implica temperaturas ≈ 121ºC a presiones de 1,2 bares, durante un tiempo variable en función del tamaño de los envases. El ahumado combina dos efectos: por un lado, el secado y por otra parte, el efecto de la temperatura, la cual aumenta hasta 121ºC alcanzando los 60ºC en el interior del producto, mientras que el tiempo dependerá del grosor de los filetes.
En productos frescos y congelados es posible amplificar fragmentos de ADN de elevado tamaño (≥1 kb), pero en el caso de productos sometidos a procesos de transformación agresivos, como los descritos previamente, esto no es posible. Distintos autores han establecido un tamaño máximo no superior a 200 pb para asegurar la amplificación del ADN en la PCR. Además, la presencia de aditivos alimentarios como
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especias o salsas, atenúan e incluso pueden llegar a inhibir totalmente las reacciones de PCR. El ADN es muy sensible a agentes ácidos y alcalinos, en este sentido, es importante destacar los productos en escabeche en los que el bajo pH favorece la degradación del ADN. Por esta razón, es muy importante definir el tamaño a amplificar en cada una de las metodologías, así como utilizar un método de extracción de ADN adecuado de tal modo que se maximice el éxito de la PCR [6, 13].
La finalidad última de este proceso de validación es garantizar que los procesos de transformación aplicados a las muestras que serán analizadas, no modifican la especificidad de los métodos de diagnóstico desarrollados, evaluado la coincidencia entre la especie identificada en base a caracteres morfológicos y la identificada con las metodologías genéticas desarrolladas.
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APLICACIONES DE LA TÉCNICA FINS
Entre las principales aplicaciones de los métodos FINS destacan las siguientes:
Identificar especies en alimentos. Evitando la sustitución parcial o total con materias primas de menor calidad y/o valor comercial en productos elaborados. Asesorar al sector transformador en materia de etiquetado, con el fin de evitar el fraude al consumidor y entre empresas del sector. Garantizar la trazabilidad de un alimento desde su origen. Esta aplicación es muy utilizada en el caso de productos de calidad con denominación de origen. Control de calidad y de importaciones. Conservación de recursos genéticos de interés alimentario. Control de pesca ilegal, no declarada y no reglamentada (INDNR).
Los principales beneficiarios de la aplicación de las herramientas moleculares son:
El sector transformador, que dispone de herramientas de control para el correcto etiquetado de productos elaborados. Las Autoridades Sanitarias y de Comercio Exterior. Consumidores en general, que gracias a la aplicación de estos métodos disponen de una mayor transparencia en los productos consumidos.
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VENTAJAS Y DESVENTAJAS DE LA TÉCNICA FINS
Aunque la secuenciación ha demostrado ser la forma más directa y fiable de obtener gran cantidad de información de los fragmentos de ADN amplificados, también es una metodología con un proceso más largo y costoso que otras técnicas. Su alta especificidad la convierten en la metodología más apropiada para la identificación de especies.
En comparación con otras técnicas, presenta las siguientes VENTAJAS:
Técnica altamente específica, sensible, precisa y reproducible. Los resultados no se ven afectados por la variabilidad intraespecífica, y permite la detección de nuevas especies no estudiadas hasta el momento. Por otro lado, posee ciertas DESVENTAJAS: El equipamiento requiere una inversión considerable. El nivel de automatización posible es bajo. Además, es necesario personal altamente cualificado para su realización. No es el método más apropiado para el análisis de muestras que contienen mezcla de especies.
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OBJETIVOS
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OBJETIVOS
Desarrollar distintos métodos identificación genética mediante la reacción en cadena de la polimerasa (PCR) seguida de análisis filogenético de las secuencias de ADN (FINS), que permita identificar las principales especies de carnes, jureles, sardinas, rayas, anguilas y gádidos. Los métodos serán aplicables a los formatos comerciales presentes en los mercados. Desarrollar herramientas moleculares basados en la técnica FINS, la detección e identificación de mezclas de especies de sardinas (Sardina pilchardus y Sardinella aurita) y de gádidos (G. morhua, G. macrocephalus y G. ogac). Evaluar los sistemas desarrollados mediante un proceso de validación metodológica. Éste permitirá comprobar que determinados ingredientes y aditivos, así como los tratamientos aplicados en el proceso de transformación, no afectan al correcto desempeño de las metodologías diseñadas. De este modo, se verificará que los métodos producen resultados válidos para los fines previstos. Evaluar la aplicabilidad de los sistemas desarrollados utilizándolos para el análisis de muestras comerciales, adquiridas en mercados españoles e internacionales y pertenecientes a alguno de los grupos estudiados (carnes, jureles, sardinas, rayas y bacalaos).
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Authentication of species in meat products by genetic techniques
European Food Research and Technology Zeitschrift für Lebensmittel- Untersuchung und -Forschung A
ISSN 1438-2377 Volume 232 Number 3
Eur Food Res Technol (2011) 232:509-515 DOI 10.1007/ s00217-010-1417-1
1 23 Your article is protected by copyright and all rights are held exclusively by Springer- Verlag. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication.
1 23 Author's personal copy
Eur Food Res Technol (2011) 232:509–515 DOI 10.1007/s00217-010-1417-1
ORIGINAL PAPER
Authentication of species in meat products by genetic techniques
Fa´tima C. Lago • Beatriz Herrero • Marı´a Madrin˜a´n • Juan M. Vieites • Montserrat Espin˜eira
Received: 29 September 2010 / Revised: 15 December 2010 / Accepted: 20 December 2010 / Published online: 9 January 2011 Ó Springer-Verlag 2011
Abstract In the present work, a method for the authen- Keywords Meat Genetic identification PCR FINS tication of meat products was developed, using Polymerase Chain Reaction (PCR) followed by Forensically Informa- tive Nucleotide Sequencing (FINS). This study describes Introduction the use of sequencing of the cytochrome b gene of the mtDNA in a wide variety of species to diagnose adultera- Meat products represent a large portion of the food tion of meat through the substitution from one species to industry. Its role in our nutrition has great importance another that have less commercial value. The main because is well known that meat products are an excellent importance of this work is to deal with a wide variety of protein and energy source for our daily diets. In a global- species that have not previously been analyzed. This ized and competitive market, the identification of food methodology strategy allows the authentication of meat species has received great attention, not only due to the species in all kind of products, fresh, or precooked prod- economic point of view but also to food allergies, medical ucts, the more usual format for marketing in that species. requirements, religious practices, and ethic reasons. This method shows a specificity of 100%. The developed Food adulteration is an important problem in the food methodology was validated and finally applied to 20 industry. This fraudulent practice is accentuated with the commercial samples including some that had been sub- aperture of frontiers and free market between countries, jected to intensive thermal treatment. In 15% of the prod- and it consists in the adulteration of the original raw ucts analyzed, the name of the species displayed in the material declared in the final product by the total or partial label was not in agreement with the identified species. The substitution of another species that have less commercial main novelty of this work lies in the fact that it allows the value [1, 2]. Some of the products most frequently related identification of a large number of meat species not ana- with food fraud are meat products, especially those that are lyzed so far in previous works. In this work are included made with meat mixtures. meat species, which can be easily found in our markets, In order to check the origin and composition of these and a wide variety of others whose consumption is com- products, the European Commission has established one mon in other parts of the world. Therefore, this technique legislation that imposes to declare the species that was used can be used as a routine method to avoid the mislabeling in in the manufacture of meat products in its labeling (CE No the marketing of these products and to assess their correct 2001/101) [3]. traceability. Furthermore, different methods based on protein and DNA analysis have been developed in order to protect consumers’ rights against these potential food fraud. Many analytical approaches have been applied for F. C. Lago B. Herrero M. Madrin˜a´n authentication of meat products. Protein-based methods & J. M. Vieites M. Espin˜eira ( ) were developed as ELISA [4], HPLC [5], electrophoresis Area of Molecular Biology and Biotechnology, ANFACO-CECOPESCA, Vigo, 36310 Pontevedra, Spain [6]. However, these methods are often unsuitable for e-mail: [email protected] complex food products, as they cannot differentiate closely 123 Author's personal copy
510 Eur Food Res Technol (2011) 232:509–515 related species in highly processed product and they are described by Roger and Bendich with slight modifications also significantly less sensitive in processed meats that [20]. The essential modifications were as follows: DNA have been put to high temperatures in their manufacturing fraction is treated with proteinase K for the same time as [7]. the CTAB buffer and followed by phenol/chloroform, In recent years, molecular authentication methodologies chloroform, and isopropanol washes. The second CTAB based on the PCR amplification have been developed, and wash and the first rehydratation with TE buffer are deleted. these methods have been successfully applied for species Only one ethanol wash is carried out. The obtained DNA authentication in meat products. These techniques are was diluted in 100 lLof19 Tris–EDTA (TE) buffer sensitive, reliable, and fast, as Restriction Fragment Length (Sigma). In the case of highly processed products, 150 mg Polymorphism (PCR–RFLP techniques) [8–11], sequenc- was used for the methodological validation and analysis of ing specific gene assay [7, 12, 13], multiplex PCR assay these commercial samples. [14–17], SNaPshot minisequencing analysis [12], and Quantity was determined by measuring the absorbance RT-PCR [18]. at 260 nm, and quality was determined by measuring the In the field of genetic identification of species in meat absorbance at 260/280 nm and 234/260 ratios [21] using a products, several studies have been carried out to date, NanoDropTM ND-1000 spectrophotometer (Thermo Sci- but all of them have some drawbacks. The main draw- entific). DNA extractions were appropriately labeled and backs in these studies are that they do not cover most of stored at -20 °C for subsequent tasks. the species present in the current international market or they have a small amount of individuals to study, so that PCR amplification and DNA sequencing intraspecific variability can exist and are not detected [7, 8, 19]. Sequences of the cytochrome b (cyt b) gene were down- In this work, FINS methodology was employed to carry loaded from the National Center for Biotechnology Infor- out a global study, which includes not only species com- mation (NCBI; Table 1). Next, these were aligned with monly consumed in our country and easy to purchase in our BioEdit 7.0 [22] and for them, a degenerate primer set was markets (cow, pig, horse, goat, sheep, rabbit, chicken, designed by hand. The name and sequence of the forward turkey, etc) but also others whose consumption is common and reverse primers are, respectively, MEAT F (50 CGA in other parts of the world (buffalo, kangaroo, ostrich, etc). GGC CTM TAY TAY GG 30) and MEAT R (50 ATT GAK Due to the globalized market, these species may be avail- CGT AGG ATT GCG TA 30). able to consumers in our markets. In all cases, the PCR were carried out in a total volume This developed methodology can be very useful in the of 50 lL with the following composition: 100–300 ng of normative control of these products, particularly in the DNA template were added to a PCR mix consisting of authentication of imported foodstuffs, in the correct 0.8 mM dNTP mix (Bioline), 5 lL109 buffer, 2 mM TM labeling, and in the protection of the consumers’ rights. MgCl2, 0.75 units of BioTaq DNA polymerase (Bioline), 0.8 lM of each primer (Sigma Genosys), and molecular biology grade water (Eppendorf) up to adjust to the final Materials and methods volume. PCR were carried out in a MyCyclerTM thermocycler Sample collection, storage (BIO-RAD). Conditions of cycling were as follows: a pre- heating step of 3 min at 95 °C, then 35 cycles (30 s at Authenticated muscle samples of species were obtained 95 °C, 30 s at 50 °C, and 30 s at 72 °C) and a final (between three and seven individuals of each species) from extension step of 72 °C for 3 min. local suppliers, universities, and research centers and were In order to ensure the proper working of PCR amplifi- used to calibrate the method developed in this research cation, PCR products were loaded in agarose gels (Sigma) (Table 1). The samples were labeled and preserved at at 2% in TBE buffer and 5 lg/mL of ethidium bromide -80 °C. (Sigma) allowing band detection. Moreover, 20 meat products were obtained in shops and DNA fragments were visualized using the Molecular markets from Spain, in order to apply the developed Imager Gel Doc XR System transilluminator and the methodology to commercial samples. software Quantity One v 4.5.2 (Bio-Rad). Size of amplified fragments was estimated from the molecular Marker DNA extraction TrackltTM100 bp DNA ladder (Invitrogen). Double-stran- ded PCR products were purified before sequencing reaction Genomic DNA was extracted from 30 mg of muscle in using NucleospinÓ 96 Extract II (Macherey–Nagel) fresh and frozen samples, according to the method according to the manufacturer’s protocol. 123 Author's personal copy
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Table 1 Species included in the present work Family Subfamily Scientific name Common name NCBI number accession
Bovidae Bovinae Bos taurus Cow FJ971083 Bubalus bubalis Water Buffalo EU760479 Bison bison Bison AF036273 Tragelaphus strepsiceros Kudu AF022063 Tragelaphus oryx Eland AF036278 Hippotraginae Oryx beisa East African Oryx DQ138199 Oryx gacella Gemsbuck AF249973 Oryx leucoryx Greek Oryx AF036286 Antilopinae Antidorcas marsupialis Springbok AF036281 Gacella dama Dama Gazelle AF025954 Antilope cervicapra Blackbuck AF022058 Alcelaphinae Connochaetes taurinus Blue Wildebeest AF016638 Connochaetes gnou Black Wildebeest AF016637 Damaliscus pygargus Bontebok AF016639 Caprinae Ovis aries Sheep NC001941 Capra hircus Goat GU068049 Aepycerotinae Aepyceros melampus Impala AF034966 Cervidae Cervinae Cervus elephus Deer EF139146 Odocoileinae Alces alces Moose AJ000026 Odocoileus hemionus Black-Tailed Deer HM222707 Odocoileus virginianus White-Tailed Deer AY607035 Rangifer tarandus Reindeer DQ673123 Equidae Equinae Equus caballus Horse AY522328 Equus asinus Ass X97337 Equus burcheri Zebra DQ470805 Suidae Sus scrofa domestica Pig AP003428 Sus scrofa Wild Boar AY634188 Camelidae Camelus bactrianus Bactrian Camel AP003423 Camelus dromedarius Dromedary AY126631 Macropodidae Macropus giganteus Eastern Grey Kangaroo AY099267 Macropus rufus Red Kangaroo U87136 Leporidae Oryctolagus cuniculus Rabbit U07566 Struthionidae Struthio camelus Ostrich U76055 Dromaiidae Dromaius novaehollandiae Emu AF338711 Anatidae Anserinae Anser anser Goose NC011196 Anatinae Anas platyrhynchos Duck FJ167857 Phasianidae Gallus gallus Chicken AF195628 Meleagris gallopavo Turkey NC_010195 Alectoris chuckar Chukar Partridge EU839475 Alectoris rufa Red-Legged Partridge AJ586184 Coturnix coturnix Quail AF112363 Perdix perdix Gray Partridge AF028791
The concentration and purity were measured by means previously in a final volume of 10 lL with BigDye Ter- of the NanoDropTM ND-1000 spectrophotometer (Thermo minator cycle sequencing ready reaction v1.1 (Applied Scientific) as described in DNA extraction. Biosystems) and sequenced on an ABI Prism 3130 (Applied Subsequently, sequencing reactions of both DNA Biosystems). Thermal cycle sequencing reaction and the strands were carried out with the primers described subsequent sequencing products’ cleanup by ethanol 123 Author's personal copy
512 Eur Food Res Technol (2011) 232:509–515 precipitation were carried out in accordance with the These products were acquired in supermarkets from Spain. manufacturer’s instructions (Applied Biosystems). Next, The main aim of these analyses was to evaluate the these sequences were analyzed with Sequencing Analysis labeling situation of these products on the market. Software v5.3.1. (Applied Biosystems) and aligned with Clustal W [23] available in the program BioEdit 7.0 [22]. The nucleotide sequences obtained were submitted to the Results and discussion GeneBank database of the National Centre for Biotech- nology Information (NCBI). Development of FINS methodology
Development of FINS methodology DNA amplification with the primers MEAT F/R generated an amplicon of 555 bp in all species included in the present A phylogenetic analysis was carried out using the software study (Table 1; accession numbers HQ122569-HQ122609; Mega 4.0 [24]. The genetic distances among the obtained Figure 1). sequences were estimated using the Tamura-Nei substitution The FINS technique (Forensically Informative Nucleo- model [25]. The inference of the phylogenetic trees was tide Sequencing) described by Bartlett and Davidson [27] carried out with the neighbor-joining method [26]. The was used in the present study to develop an identification reliability of the clades formed at the species level in the trees method for meat species. This technique is based on the was evaluated by means of a bootstrap test. The degree of comparison between sequences pattern species and confidence assigned to the nodes in the phylogenetic trees sequences of unknown samples. was estimated by bootstrapping with 2,000 replicates. Genetic distances between cyt b gene sequences obtained of all studied species were estimated using the Methodological validation Tamura-Nei method [25]. The phylogenetic analysis of the fragment amplified The main commercial treatments types were applied to allows to establish the relationships among species by tissue of different species. These treatments included fresh, means of the construction of phylogeny using the data sets. frozen, boiling, pickling, salting, smoking, battering, In the tree obtained, all the sequences belonging to indi- breading, precooking, and minced meats with different viduals of the same species were grouped in the same kinds of sauces and condiments. cluster, allowing their identification. Two strongly sup- One of the most aggressive treatments applied is the ported clades were identified: Mammalia and Aves, which smoking that combines two effects: on the one hand, corresponded with the two Class included in this work. salting and drying steps and on the other, the effect of high Bootstraps values of branches at level species were higher temperatures. To smoking the fillets, the temperature was than 97, reflecting the robustness of the phylogenetic tree raised to 121 °C and inside the product it reached 60 °C. (Figure 2). The cooking time depended on the thickness of the fillets. All these treatments were carried out in the pilot plant of CECOPESCA (Spanish National Centre of Fish Processing Technology). Two processed products for each one of the kind of manufacturing processes for every species were included in this study, for each of which different kinds of sauces and condiments were used. Next, the products were analyzed with the methodology developed in the present work. Results of the species assignment on the basis of morphology and genetic probes were compared. The coincidence percentage between the species identified on the basis of morphological traits, and the genetic methodology developed was calculated to establish the specificity of the method.
Application to commercial products
After the validation of the methods developed in the Fig. 1 PCR products of processed and non-processed meat samples present work, these were applied to 20 meat products. obtained in the PCR (Size obtained: 555 bp) 123 Author's personal copy
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Family Class
100 Aepyceros melampus (Impala) 100 Antidorcas marsupialis (Springbok) 100 Antilope cervicapra (Blackbuck) 100 Gacella dama (Dama gazelle) 100 Damaliscus pygargus (Bontebok) 100 Connochaetes taurinus (Blue wildebeest) 100 Oryx leucoryx (Greek orux) 100 Oryx gazella (Gemsbuck) Bovidae 100 Oryx beisa (East african oryx) º 100 Bos taurus (Cow) 100 Bison bison (Bison) 100 Bubalus bubalis (Water buffalo) 100 Tragelaphus strepsiceros (Kudu) 100 Tragelaphus oryx (Eland) 100 Capra hircus (Goat) Mammalia 100 Ovis aries (Sheep) 97 100 Odocoileus virginianus (White-tailed deer) 97 Odocoileus hemionus (Black-tailed deer) Cervidae 100 Alces alces (Moose) 100 Rangifer tarandus (Reindeer) 100 Cervus elephus (Deer) 100 Sus scrofa Suidae 100 Camelus dromedariu (Dromedary) 100 Camelus bactrianus (Bactrian camel) Camellidae 100 Equus caballus (Horse) 100 Equus asinus (Ass) Equidae 100 Equus burchellii (Zebra) 100 Oryctolagus cuniculus (Rabbit) Leporidae 100 Macropus giganteus (Eastern grey kangaroo) 100 Macropus rufus (Red kangaroo) Macropodidae 100 Struthio camelus (Ostrich) Struthionidae 100 Dromaius novaehollandiae (Emu) Dromaiidae 100 Anser anser (Goose) Anatidae 100 Anas platyrhynchos (Duck) 100 Perdix perdix (Gray partridge) 100 Meleagris gallopavo (Turkey) Aves 100 Gallus gallus (Chicken) Pnasianidae 100 Coturnix coturnix (Quail) 100 100 red-leggedAlectoris partridge rufa (Red-legged partridge) 97 Alectoris chukar (Chukar partridge)
0.05
Fig. 2 Phylogenetic tree showing the relationships among the studied meat species, carried from the alignment of 555 bp of the cyt b gene
In this work, the most common species of interest Phasianidae. One can see that all the subfamilies are well commercial have been included. The phylogenetic analysis grouped and differentiated from each other. shows seven families belonging to Class Mammalia: The technique developed allowed the unequivocal Bovidae, Cervidae, Equidae, Suidae, Leporidae, Macro- identification of all the meat species included in this work. podidae, and Camelidae. The most represented family is the Bovidae. This family is composed for nine subfamilies. Methodological validation In this study are represented six different subfamilies: Aepycerotinae, Antilopinae, Alcelaphinae, Hippotraginae, The aim of the methodological validation is to check Bovinae, and Caprinae. whether the manufacturing processes that receive the pro- Also have been included four families belonging to the cessed food have not any influence on the identification of Class Aves: Struthionidae, Dromaidae, Anatidae, and these species.
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Different products were prepared in the pilot plant of products. In 15% of the products analyzed, the name of the CECOPESCA simulating the conditions used in the food species displayed in the label was not in agreement with the industry. This approach is useful to assess the functioning identified species, as determined by genetic analysis using and optimize the conditions of the developed methodology. the methodology herein developed (Table 2). Standard individuals were subjected to several transfor- mation processes, allowing evaluating the influence of different variables on the genetic method here proposed. Conclusion Species identified from these samples by means of the FINS method herein developed were in agreement with This article describes a DNA-based method that allows the those identifications obtained on the basis of morphological genetic identification of meat species in fresh and pro- characters. This method shows a specificity of 100%. cessed products. It is worth highlighting that this method is the most completely developed one to date in terms of the Application to commercial products number of species included. Among the advantages of this technique, it is worth highlighting is its specificity of Although adulteration and substitution deliberate or unin- 100%. tentional to which meat products are exposed, there are few Main applications of this methodology are normative studies that reveal these data. There are studies that doc- control of raw and processed products, particularly the umenting the existence of mislabeled products as is the authentication of imported species; verification of the case of adulteration of sausages labeled as ostrich that had traceability of different meat batches along the commercial been adulterated with pork meat or highly processed meat chain; control of the correct labeling; and protection of the products labeled as turkey, which had been replaced by consumer’s rights. chicken [11]. In this work, the methodologies developed were applied Acknowledgment We thank Rod Asher (Cawthron Institute, Nel- to 20 meat products including different levels of transfor- son, New Zealand), Peter Hishon (New Zealand Ostrich Export Corporation, Alexandra), and Arthur Crimp (Wakefield) for providing mation. This application allowed to know the degree of some of the samples included in this work. fulfillment of the labeling regulations in these meat
References
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Genetic Identification of Horse
Mackerel and Related Species in
Seafood Products by Means of
Forensically Informative Nucleotide
Sequencing Methodology
Página 63 de 153
ARTICLE
pubs.acs.org/JAFC
Genetic Identification of Horse Mackerel and Related Species in Seafood Products by Means of Forensically Informative Nucleotide Sequencing Methodology F atima C. Lago, Beatriz Herrero, Juan M. Vieites, and Montserrat Espi~neira* Area of Molecular Biology and Biotechnology, ANFACO-CECOPESCA, Vigo, 36310 Pontevedra, Spain
ABSTRACT: In the present study, a methodology based on the amplification of a fragment of mitochondrial cytochrome b and subsequent phylogenetic analysis (FINS: forensically informative nucleotide sequencing) to genetically identify horse mackerels have been developed. This methodology makes possible the identification of more than 20 species belonging to the families Carangidae, Mullidae, and Scombridae. The main novelty of this work lies in the longest number of different horse mackerel species included and in the applicability of the developed methods to all kinds of processed products that can be found by consumers in markets around the world, including those that have undergone intensive processes of transformation, as for instance canned foods. Finally, the methods were applied to 15 commercial samples, all of them canned products. Therefore, these methods are useful for checking the fulfillment of labeling regulations for horse mackerels and horse mackerel products, verifying the correct traceability in commercial trade, and fisheries control. KEYWORDS: Horse mackerel, Trachurus, Carangidae, genetic identification, FINS, PCR
’ INTRODUCTION require the need to create a methodology for rapid and accurate fi Horse mackerels belong to the family Carangidae. This group identi cation. Molecular techniques arise in response to this fi of small pelagic fish, which includes about 30 genera and approx- need, allowing the authentication of the nal product, avoiding imately 140 species, is well represented in all tropical and sub- unintended substitutions or fraud in the labeling and rigorous tropical seas. The main characteristics of this taxon are its control of the species caught. aerodynamic shape, laterally compressed body, slender tail base, In this sense, molecular techniques based on DNA analysis are and a strongly forked caudal fin. the best option as they provide more genetic information. In The properties of their meat, which is tasty but not overly juicy addition, mitochondrial DNA has certain characteristics (high and has a high nutritional value, makes most horse mackerels mutation rate, small genome size, maternal inheritance, lack of highly valued for human consumption, which positions it as a recombination, and high number of copies per cell) that will pro- very important pelagic resource in artisanal and industrial fish- vide numerous advantages over nuclear DNA, making it an eries around the world. Preferably, they are sold whole, fresh, excellent marker for this type of analysis. In turn, cytochrome b chilled, or frozen, but they also can be found for sale gutted or (cyt b) mitochondrial also presents optimal characteristics (slow evolution, conserved sequence, low intraspecific variation, and beheaded, filleted, and ready to cook skinless and boneless. They high interspecific variation) that has led to its use as a molecular are also sold dried, salted, and smoked, and small size mackerels marker in numerous studies of species belonging to the - are intended for the canning industry, mainly in oil or pickled. Carangidae family 6 8 and other studies of genetic identification of fi - When they are sold whole, their identi cation at the species level different taxonomic groups.9 15 To date, there have been many can be possible through their morphological features; in other works based on molecular techniques to identify different species cases, the mentioned characteristics are not present, hindering of horse mackerels. Most of them are based on analysis of mito- fi and preventing their identi cation. chondrial DNA fragments through polymerase chain reaction- - On the other hand, because of high demand of this resource, restriction fragment length polymorphism (PCR-RFLP)16 18 or fi horse mackerel sheries are continually threatened by over- SNP.19 They all have great disadvantages that include few exploitation. In the case of Chilean jack mackerel (Trachurus species. All of these studies only include the Mediterranean murphyi), the large number of captures along with the high rate of species (Trachurus mediterraneus, Trachurus trachurus, and Tra- migration and the seasonality of the species has made this churus picturatus) and, in some cases, included two species of the resource undergo a critical situation marked by a decline in its genus Mullus.19 For these reasons, in this paper has been devel- stock by 80% in the last 15 years.1,2 This requires the existence of oped a methodology based on the amplification of a fragment of appropriate management of this resource, which takes into mitochondrial cyt b by means of PCR and subsequent phylogenetic account the minimum size 3 and catch volumes, with the need to update existing legislation to date that regulates the catch of Received: November 23, 2010 this pelagic species.4,5 Accepted: January 5, 2011 Into a globalized market, both the high demand for horse Revised: December 29, 2010 mackerels and the severe shortages that cross some of the species Published: February 18, 2011
r 2011 American Chemical Society 2223 dx.doi.org/10.1021/jf104505q | J. Agric. Food Chem. 2011, 59, 2223–2228 Journal of Agricultural and Food Chemistry ARTICLE
Table 1. Species Included in the Present Work
family scientific name common name samplesa locationb NCBI
Trachurus capensis Cape horse mackerel 3 AT, ZAF AY526536 Trachurus declivis Jack mackerel 4 AUS, NZ, TAS AY526542 Trachurus delagoa African scad 2 ZAF Trachurus indicus Arabian scad 2 IND Trachurus japonicus Jack mackerel 3 JAP AB018994 Trachurus lathami Rough scad 2 CAN, USA AF363748 Trachurus mediterraneus Mediterranean horse mackerel 3 ESP, GRE EU224037 Trachurus murphyi Inca scad 7 PER, CHI, ARG, AUS Trachurus novaezelandiae Yellowtail horse mackerel 2 AUS AY526545 Trachurus picturatus Blue jack mackerel 3 MOR, AT, M EF439614 Trachurus symmetricus Pacific jack mackerel 3 USA, MEX AY526541 Carangidae Trachurus trachurus Atlantic horse mackerel 10 ESP, M, POR EU224040 Trachurus trecae Cunene horse mackerel 2 NAM, MOR AY050740 Caranx caballus Green jack 2 USA AY050721 Caranx crysos Blue runner 2 AT, M EF392575 Caranx hippos Crevalle jack 3 AT, M AY050720 Caranx sexfasciatus Bigeye trevally 2 IND Carangoides ferdau Blue kingfish 2 IND Decapterus macrosoma Scad 3 IND, PER Pseudocaranx dentex Silver travally 3 ESP EF392607 Selar crumenophthalmus Bigeye scad 2 PER Uraspis secunda Cottonmouth jack 2 ZAF Mullidae Mullus barbatus Red mullet 2 UK, ESP EU036452 Rastrelliger kanagurta Indian mackerel 3 IND Scombridae Rastrelliger faughni Island mackerel 2 IND a Between 3 and 10 individuals were studied by each sample. b Location abbreviations: ARG, Argentina; AT, Atlantic; AUS, Australia; CAN, Canada; CHI, China; ESP, Spain; GRE, Greece; IND, India; JAP, Japan; M, Mediterranean Sea; MEX, Mexico; MOR, Morocco; NAM, Namibia; NZ, New Zealand; PER, Peru; POR, Portugal; TAS, Tasmania; UK, United Kingdom; USA, United States; and ZAF, South Africa.
analysis. Unlike the studies published to date, this technique described and subsequent purification by means of the Nucleospin allows us to genetically identify over 20 species of horse mackerels Extract II kit (Macherey-Nagel) following the supplier's protocol. that can be found by consumers in markets around the world in The quality and quantity were determined by measuring the absor- its different forms of marketing. Besides the advantages already bance at 260 nm and the 260/280 nm and 234/260 ratios 23 using a mentioned, the development of these methodologies has the NanoDrop ND-1000 spectrophotometer (Thermo Scientific). DNA final aim of improving fisheries policy, ensuring compliance with extractions were appropriately labeled and stored at -80 °C for subse- current legislation as far as catch and size, and preserving quent tasks. endangered stocks. Primers Design, PCR Amplification, and DNA Sequencing. Sequences of the cyt b gene were downloaded from the National Center for Biotechnology Information (NCBI) (Table 1). These were aligned ’ MATERIALS AND METHODS with BioEdit 7.0,24 and for them, a degenerate primer set was designed by hand. The name and sequence of the forward and reverse primers are, Sample Collection and Storage. Samples of different horse respectively, JUREL F, 50-CAC GAA ACM GGV TCC AAC AA-30, and mackerel and related species were collected from several locations JUREL R, 50-ATG GCR TAK GCA AAB AGG AA-30 . around the world (Table 1). When it was possible, the individuals were In all cases, the PCR reactions were carried out in a total volume of 20,21 identified according to morphological characteristics. In other cases, 50 μL with the following composition: 100-300 ng of DNA template ethanol-preserved fish tissues were provided by universities and research was added to a PCR mix consisting of 0.8 mM dNTP mix (Bioline), 5 μL ff centers located around the world. Once identified, samples were labeled of 10 bu er, 2 mM MgCl2, 0.75 units of BioTaq DNA polymerase and preserved at -80 °C. Moreover, 15 canned products labeled as (Bioline), 0.8 μLofa10μM solution of each primer (Sigma Genosys), horse mackerel were provided by import industries or purchased in and molecular biology grade water (Eppendorf) to adjust to the final supermarkets and shops from Europe, to apply the developed metho- volume. dology to commercial samples (Table 2). Polymerase chain reactions were carried out in a MyCycler thermo- DNA Extraction. Genomic DNA was extracted from 30 mg of cycler (BIO-RAD). Conditions of cycling were as follows: a preheating muscle in fresh and frozen samples, according to the method described step at 94 °C for 5 min, 35 cycles of amplification (95 °C for 20 s, 58 °C by Roger and Bendich with slight modifications.22 The obtained DNA for 20 s, and 72 °C for 20 s), and a final extension step of 72 °C for 7 min. was diluted in 100 μLof1 Tris-EDTA (TE) buffer (Sigma). In the PCR amplicons were visualized on 2% agarose gels (Sigma) in 1 TBE case of products used for the methodological validation and commercial buffer (Sigma) with 0.3 μg/mL of ethidium bromide (Sigma). DNA samples from amounts of tissue comprising between 100 and 300 mg, fragments were visualized using the Molecular Imager Gel Doc XR Sys- the extraction of DNA was carried out by the CTAB method previously tem transilluminator and the software Quantity One_ v 4.5.2 (BIO-RAD).
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Table 2. Commercial Samples Analyzed with the Methodology Developed
canned products samples labeled species identified species sample codea
natural natural in water 1 Horse Mackerel/Chinchards T. murphyi S1
oils olive oil 1 Horse Mackerel/Chinchards T. murphyi S2 3 Horse Mackerel/Chinchards T. trachurus S3-S5 sunflower oil 2 Horse Mackerel/Chinchards T. murphyi S6-S7 2 Horse Mackerel/Chinchards T. trachurus S8-S9 soya oil 1 Horse Mackerel/Chinchards T. trachurus S10 sauces tomato sauce 1 Horse Mackerel/Chinchards T. murphyi S11 1 Horse Mackerel/Chinchards T. trachurus S12 curry sauce 1 Horse Mackerel/Chinchards T. trachurus S13 pickled sauce 2 Horse Mackerel/Chinchards T. trachurus S14-S15 a Code shown in Figure 2 that locates the commercial samples in the phylogenetic tree of the studied species.
The 50 Base Pair Ladder DNA marker (Tracklt, Invitrogen) was used to ’ RESULTS AND DISCUSSION estimate the size of the amplicons. Next, double-stranded DNA was purified using the Nucleospin 96 PCR Amplification. Obtained amplification and sequencing Extract II (Macherey-Nagel) according to the manufacturer's instruc- PCR products and DNA amplification with the primers JUREL tions. The concentration and purity were measured by means of the F/R generated an amplicon of 239 bp in all of the species Nano-DropTM ND-1000 spectrophotometer (Thermo Scientific) as included in the present study (Table 1) (accession numbers described for DNA extraction. Subsequently, PCR products were HQ593670-HQ593727). The main objective of the design of sequenced with the primers used for amplification in an automatic primers by hand is the fact that in this way it is possible to make a DNA Genetic Analyzer (ABI Prism 3130 Genetic Analyzer) using the thorough analysis of conserved mitochondrial DNA regions in all BigDye Terminator cycle sequencing kit v.1.1 (Applied Biosystems) species under study. In this way, we can design primers that allow following the manufacturer's recommendations. Raw data were analyzed one to differentiate all of these species. using the Sequence Analysis software v.5.3.1. (Applied Biosystems). In the case of fish that have undergone different treatments The sequences were analyzed with the Chromas 1.45 software25 and 26 24 such as canned, it is not possible to amplify PCR products of a aligned with Clustal W available in the program BioEdit 7.0. The large size, because the thermal treatment generates DNA frag- nucleotide sequences obtained were submitted to the GeneBank data- mentation. This was the case for the canned products, where base of the National Centre for Biotechnology Information (NCBI). fi Development of FINS Methodology. Phylogenetic analyses fragments of little sizes were ampli ed. Quinteiro et al. estab- - lished a maximum fragment size in canned products of 176 bp to were carried out using the software Mega 4.0 using the Tamura Nei 30 27 ensure the amplification. Other authors, under certain condi- model to calculate the genetic distances between sequences. The fi inference of the phylogenetic tree was carried out with the neighbor- tions, ampli ed fragments higher than 250 bp from canned 11,31-34 joining method.28 The reliability of the clades formed at the species level products. However, we consider that it is important to in the tree was evaluated by means of a bootstrap test with 2000 repli- have at one's disposal a method that can be used routinely, cations. Also, the MEGABLAST search available at NCBI was assessed allowing the easily amplification of DNA and providing a reliable to assign the sequences to a particular species.29 species identification. Herein, the amplification of a small frag- Methodological Validation. Individuals from different species ment is proposed that contains enough information to enable the were authenticated on the basis of their morphological traits, and main differentiation of all of the studied species. commercial treatment types were applied to them. Treatments included The presence of additives used in the alimentary industry as canning, fresh, and frozen for each one of the different kinds of sauces, spices or sauces attenuates or inclusively inhibits the DNA ampli- and condiments were used. fi ff ° cation. Moreover, the di erent kinds of sauces added produce The treatment applied to canned samples involved 121 C tempera- differences in the quantity and quality of the extracted DNA as ture and 1.2 bar of overpressure, and the time varied depending on the this molecule is very sensitive to acid and alkaline agents. In this size of the can. All of these treatments were carried out in the pilot plant sense, it is worth highlighting the pickled products, in which the of CECOPESCA (Spanish National Centre of Fish Processing Tech- low pH produces higher DNA degradation. For this reason, the nology). Next, products were analyzed with the methodology developed fi in the present work. The coincidence percentage between identified strategy of ampli cation proposed facilitates the successful PCR fi 11 species on the basis of morphological traits and the genetic methodology ampli cation in any seafood product. developed was calculated to establish the specificity of the method. Development of Forensically Informative Nucleotide Sequencing (FINS) Methodology. The FINS technique de- Application to Commercial Products. After the validation of 35 the methods developed in the present work, these were applied to 15 scribed by Bartlett and Davidson was used in the present study canned products labeled as horse mackerel (Table 2). These products to develop an identification method for horse mackerel and were acquired in supermarkets from Europe. The purpose of these related species. Sequences of unknown samples are compared analyses was to evaluate the situation regarding the labeling of these with sequences belonging to pattern specimens on the basis of products on the market. this technique.
2225 dx.doi.org/10.1021/jf104505q |J. Agric. Food Chem. 2011, 59, 2223–2228 Journal of Agricultural and Food Chemistry ARTICLE
Figure 1. Distribution map of the horse mackerel and related species included in the present study.
The genetic distances between the cyt b gene sequences of all available at NCBI was assessed to assign any horse mackerel and of the studied species were estimated using the Tamura-Nei related species DNA sequences to a particular species. The method.36 The phylogenetic analysis of the amplified fragment phylogenetic assignments generated by the proposed FINS tech- (199 bp without primers) was carried out, allowing establish- nique were compared to the results obtained by BLAST, and the ment of the relationships among species by means of the con- same species assignments were obtained (data not shown). It is struction of phylogeny using the data set. In the tree obtained, all worth highlighting that for many species found that their of the sequences belonging to individuals of the same species sequences included into NCBI had not been properly assigned were grouped in the same cluster, allowing their identification. with the correct species. Therefore, these two techniques could The phylogenetic tree contains species from three families be used to identify horse mackerels and related species herein belonging to order Perciformes: Carangidae, Mullidae, and studied. Scombridae. In the Carangidae family, it is worth highlighting Methodological Validation. The aim of the methodological the clade of genus Trachurus that is a strongly supported clade validation was to check whether the manufacturing process by with a bootstrap value of 99 (Figure 2). The results obtained in which the processed foods had undergone had not influenced the this clade agree with the studios carried out by Bektas et al. and identification of these species. Different products were prepared Karaiskou et al.7,8 Both coincide in the fact that genetic distances in the pilot plant of CECOPESCA, simulating the conditions between the species indicated that T. mediterraneus and used in the food industry (Table 2). This approach is useful to T. picturatus are more closely related than T. trachurus. The boot- assess the function and optimization of the conditions of the strap method can be used to obtain the support of the different developed methodology. The standard individuals were sub- groups obtained in the phylogenetic tree. It has been calculated jected to several transformation processes, allowing evaluation of that bootstrap values higher or equal to 70 usually correspond to the influence of different variables on the genetic methods herein a probability higher or equal to 95% that the corresponding proposed. cluster is real,37 giving a quantitative measurement of the cer- Identified species in these samples by means of the method tainty of the assignment of a sample to a particular species. The herein developed were in agreement with those based on mor- phylogenetic tree constructed from 199 bp sequences shows that phological characters. Therefore, the methodology shows a all of the sequences belonging to individuals of the same species specificity of 100%. are grouped in the same cluster (Figure 2). All clusters are stron- Application to Commercial Products. The developed gly supported, with bootstrap values higher than 70, allowing the methodology was applied to 15 canned products labeled as horse reliable assignation of each individual to a particular species. mackerel. This application allowed knowledge of the degree of Therefore, the proposed strategy of amplification allows the fulfillment of the labeling regulations in these seafood products. amplification of a DNA fragment sufficiently long to discriminate It is worth highlighting that any of tested cans were labeled with successfully all of the horse mackerel and related species of the scientific name of the species. All were labeled with the commercial importance, even in canned products and others commercial denomination of the species as Horse Mackerel or formats in which the DNA is highly degraded. Chinchard. This is a major problem in all products made from BLAST analysis is a suitable technique to find regions of local horse mackerel, as this family includes a large number of very similarity between sequences and can even be a suitable tech- similar morphologically species but not all have the same nique to identify species. Specifically, the MEGABLAST search commercial value. Obtained results showed that the most
2226 dx.doi.org/10.1021/jf104505q |J. Agric. Food Chem. 2011, 59, 2223–2228 Journal of Agricultural and Food Chemistry ARTICLE
Figure 2. Neighbor-joining tree showing the relationships among the studied species, carried from the alignment of 239 bp of the cyt b gene (fragment of 199 bp without primers).The S1-S15 codes belong to the commercial samples analyzed. common species used in canning are T. murphyi and T. trachurus India), Jose A. Gonz alez (ICCM, Spain), Sean Fennessy (Table 2). (Oceanographic Research Institute, South Africa), Kim Smith In conclusion, in the present work, one DNA-based method (Estuarine & Inshore Fisheries Australia), Tracey Fairweather that allowed the genetic identification of the most important (Marine and Coastal Management, South Africa), Konstantinos commercialized species of horse mackerel and related species has Triantafyllidis (University of Thessaloniki, Greece), Aland Connell been developed. The main advantage of this method as com- (South Africa), Jorge Castillo Pizarro (Instituto de Fomento pared to the ones published up to date is that besides including a Pesquero, Chile), Dianne J. Bray (Museum Victoria, Australia), high number of species (25 species), it is based on the use of a H. J. Walker Jr. (Scripps Institution of Oceanography, United fragment with a size below 250 bp and can be applied to all kinds States), Katherine Maslenikov (University of Washington Fish of processed products, including those that have undergone in- Collection, United States), Sahar Mehanna (National Institute tensive transformation processes. The possible applications of of Oceanography and Fisheries, Egypt), and Grigorius Krey this method are the following: normative control of raw and (Fisheries Research Institute, Greece) for kindly supplying the processed products, particularly the authenticity of imported horse mackerel and related species samples. This work was funded species; verification of the traceability of different fishing batches by the European Fisheries Fund (EFF) and the Ministerio de along the commercial chain; correct labeling; protection of the Madio Ambiente y Medio Rural y Marino (Secretaría General del consumer's rights; fair competence among fishing operators; and Mar) under the order ARM/1193/2009 of the 6th of May. fisheries' control. ’ REFERENCES ’ AUTHOR INFORMATION (1) IFOP. Instrumento Financiero de Orientaci on de la Pesca (IFOP). Corresponding Author Informe Final: Investigacion Evaluacion de Stock y CTP Jurel, 2010. (2) C ardenas, L.; Silva, A. X.; Magoulas, A.; Cabezas, J.; Poulin, E.; *Tel: (34)986 469 301. Fax: (34)986 469 269. E-mail: montse@ Ojeda, F. P. Genetic population structure in the Chilean jack mackerel, anfaco.es. Trachurus murphyi (Nichols) across the South-eastern Pacific Ocean. Fish. Res. 2009, 100 (2), 109–115. ’ ACKNOWLEDGMENT (3) Reglamento (UE) No. 53/2010 del Consejo de 14 de enero de 2010 por el que se establecen, para 2010, las posibilidades de pesca para We thank Rod Asher (Marine Biology Cawthron Institute, determinadas poblaciones y grupos de poblaciones de peces, aplicables New Zealand), Vaseeharan Baskaralingam (Alagappa University, en aguas de la UE y, en el caso de los buques de la UE, en las dem as aguas
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FINS methodology to identification of sardines and related species in canned products and detection of mixture by means of SNP analysis
systems
Página 71 de 153
Eur Food Res Technol (2011) 232:1077–1086 DOI 10.1007/s00217-011-1481-1
ORIGINAL PAPER
FINS methodology to identification of sardines and related species in canned products and detection of mixture by means of SNP analysis systems
Fa´tima C. Lago • Beatriz Herrero • Juan M. Vieites • Montserrat Espin˜eira
Received: 31 January 2011 / Revised: 31 March 2011 / Accepted: 9 April 2011 / Published online: 1 May 2011 Ó Springer-Verlag 2011
Abstract The sardines are a resource of great importance applied in questions related to correct labeling, traceability, in the artisanal and industrial fisheries worldwide. Such fishery regulations, and commercial trade control of sar- sardines and sardine-type products are included many dines and sardine-type products. species of small pelagic species, all belonging to the Clu- peidae family. Within this family, highlights the European Keywords Sardine Sardine-type products Genetic sardine (Sardina pilchardus), which by its organoleptic identification FINS PCR characteristics this species has gained an extraordinary commercial significance. In this work, an amplification of a fragment of mitochondrial cytochrome b marker and sub- Introduction sequent phylogenetic analysis (FINS: Forensically Infor- mative Nucleotide Sequencing) were carried out to assure Such sardines and sardine-type products are included many the correct labeling of sardine and sardine-type products. species, all belonging to the Clupeidae family. This family On the other hand, a single nucleotide polymorphism of small pelagic species, which encompassing more than 60 (SNP) analysis that allows detection of mixture of S. pil- genera, is well represented in warm and surface waters chardus and S. aurita in canned products was developed. worldwide. After the application of this methodology to more than 80 This group is one of the most popular fish to be one of available commercial samples can conclude that in more of the most energetic blue fish and with more vitamins and 15% of the products analyzed, the name of the species minerals. It has high fat content, which enhances the flavor displayed in the label was not in agreement with the and aroma of his flesh, and high omega-3 fatty acids con- identified species, also a one of these included in mixture tent, which reduce the incidence of cardiovascular disease. of species. The main novelty of this work lies in the fact Therefore, the sardines are one of the most valued small that have been included a long number of different species pelagic fish for human consumption, which positions it as a of sardines, many of which were not included in similar resource of great importance in the artisanal and industrial works up to date. Furthermore, this methodology allows fisheries worldwide. Within this family, in the northeast identifying over 20 species of sardines and can be applied Atlantic, highlights the European sardine or common sar- to all kinds of processed products, including those who dine (Sardina pilchardus), which by its organoleptic have been subjected to intensive processing treatments, characteristics this species has gained an extraordinary such as canned foods. Therefore, this molecular tool can be commercial significance [1, 2]. The countries of the world with more sardine production are for species belonging to Sardinops genus: Mexico (133,247 t), Thailand (47,572 t), Ecuador (27,026 t), Bra- zil (21,858 t), and Philippines (13,444 t); For Sardina pil- & F. C. Lago B. Herrero J. M. Vieites M. Espin˜eira ( ) chardus: Morocco (188,393 t), Letonia (93,940 t), Spain Area of Molecular Biology and Biotechnology, ANFACO-CECOPESCA, Vigo, 36310 Pontevedra, Spain (29,806 t), Portugal (18,350 t), and Ucrania (17,500 t) e-mail: [email protected] [FISHSTAT of FAO (2008)]. 123 1078 Eur Food Res Technol (2011) 232:1077–1086
Due to the large number of genera that includes this characteristic profiles for each species will be more com- family, and in order to improve the provisions on product plicated. In this case, it is worth highlighting that the labeling for sardines and related species of sardine’s can- analysis of a sample belonging to one of the 13 remaining ned products arises the Codex Alimentarius Stan 94 [3]. species included in the Codex Stan 94 and which has not The main objective of this standard is preventing the unfair been included in this study could generate an unexpected competition and the risk of consumer confusion that can restriction patterns, and prevent the identification or even arise with the marketing of products, all called ‘‘canned generate a misidentification of the sample. In the second sardines’’, which include various species. To this end, paper [5] describes a FINS method that uses different sets reserves the name ‘‘sardines’’ exclusively to the species of primers, which is difficult and becomes more expensive S. pilchardus, while the remaining 21 species accepted by technique. Both studies have the major drawback to include the standard should become commercialized in canned low number of species. These methodologies are now under the trade name of ‘‘X sardines’’ where X represents, obsolete in terms of number of species since the last according to the laws of each country, the species, the revision of Codex Stan 94 made in 2007 which includes common name for this species, country, geographic area, or new species that can be marketed under the name canned a combination of these data. sardines. The closest species to S. pilchardus which can more For these reasons, in this paper has developed a meth- easily lead to error are round Sardinella (Sardinella aurita) odology based on the amplification of a fragment of and European Sprat (Sprattus sprattus). These small pela- mitochondrial cyt b marker by means of PCR and sub- gic species can be available to consumers in different sequent phylogenetic analysis. Unlike the studies published formats, these can became commercialized mainly fresh to date, this technique allows to genetically identify more and whole or without head and eviscerated in preserves, but than 20 species of sardines which can be found by con- also salted, smoked or pickled. Except when they are sold sumers in markets around the world in its different forms of whole, when identifying of the species is relatively easily marketing. In addition, a SNP analysis of S. pilchardus and done through morphological features, in canned products S. aurita was also made, allowing to identify 20 different these features are not present, which makes difficult or polymorphic nucleotidic positions. Based on these SNP, a prevent their identification. methodology that allows to detect mixing of these two In the context of a globalized market which are char- species in canned products was designed. acterized by high demand of sardines throughout the year, arises the need to create a precise identification method- ology, which allows providing to the consumer of con- Materials and methods clusive information on the product purchased. To date, have been published many works based on Sample collection, storage molecular techniques to identify different species of sar- dines. Of all of them, those that use molecular techniques Samples of different sardine and related species were col- based on amplification and sequencing of a DNA fragment lected from several locations around the world (Table 1 are that offer a better response to this need, because pro- and Fig. 1). When it was possible, the individuals were viding more genetic information. Between them emphasize identified according to morphological characters [6]. In published works by Jerome et al. in 2003 [4, 5] in which a other cases, ethanol-preserved fish tissues were provided fragment of mitochondrial cyt b is studied because has by Universities and research centers located around the certain characteristics such as slow evolution, conserved world. Once identified, samples were labeled and preserved sequence, low intraspecific variation, high interspecific at -80 °C. variation, which makes it an excellent marker for studies of Moreover, 83 canned products labeled as sardine were genetic identification. In the first study [4] that describes a provided by import industries or purchased in supermarkets PCR–RFLP method which allows differentiating S. pil- and shops from Europe, in order to apply the developed chardus of eight other species. As Jerome et al. [5], we methodology to commercial samples. agree that the FINS analysis is more appropriate for the genetic identification of sardines and sardine-type products DNA extraction because the PCR–RFLP technique mentioned above does not provide a characteristic profile for each of the species Genomic DNA was extracted from 30 mg of muscle in included in this work, allow differentiating only of S. pil- fresh and frozen samples, according to the method chardus of other 8 species. Moreover, is important to have described by Roger and Bendich with slight modifications note that if species included in the study are increased, will [7]. The obtained DNA was diluted in 100 lLof19 Tris– make this technique more expensive, and obtain EDTA (TE) buffer (Sigma). In the case of products used 123 Eur Food Res Technol (2011) 232:1077–1086 1079
Table 1 Species included in the present work Family Scientific name Common name Samplesa Location
Ambligaster sirm Spotted sardinella 3 IOE, PWC Clupea harengus Atlantic herring 3 ANE, MBS Ethmalosa fimbriata Bonga shad 3 AEC Etrumeus teres Red-eye round herring 6 ASE, IOE, PEC, PNW, PWC Gilchristella aestuaria Gilchrist’s round herring 3 ASE Herklotsichthys quadrimaculatus Bluestripe herring 3 IOE, PWC Hyperlophus vittatus Sandy sprat 3 PSW Nematolosa vlaminghi Western Australian gizzard shad 3 IOE Ophistonema bulleri Slender thread herring 3 PEC Clupeidae Ophistonema medirastre Middling thread herring 3 PEC Sardina pilchardus European pilchard 6 AEC, ANE, MBS Sardinella albella White sardinella 3 IOE, PWC Sardinella aurita Round sardinella 6 AEC, ANW, ASW, AWC, MBS Sardinella longiceps Indian oil sardine 3 IOW Sardinella maderensis Madeiran sardinella 3 AEC, MBS Sardinops melanostictus South American pilchard 3 PNW, PWC Sardinops sagax South American pilchard 4 ASE, IOE, PEC, PWC Sprattus sprattus European sprat 3 ANE Strangomera bentincki Araucanian herring 3 PSE Pristigasteridae Ilisha africana West African ilisha 1 AEC Ilisha elongata Elongate ilisha 1 PWC AEC Atlantic, Eastern Central (FAO 34), ANE Atlantic, Northeast (FAO 27), ANW Atlantic, Northwest (FAO 21), ASE Atlantic, Southeast (FAO 47), ASW Atlantic, Southwest (FAO 41), AWC Atlantic, Western Central (FAO 31), IOE Indian Ocean, Eastern (FAO 57), IOW Indian Ocean, Western (FAO 51), MBS Mediterranean and Black Sea (FAO 37), PEC Pacific, Eastern Central (FAO 77), PNW Pacific, Northwest (FAO 61), PSE Pacific, Southeast (FAO 87), PSW Pacific, Southwest (FAO 81), PWC Pacific, Western Central (FAO 71) a Between 3 and 10 individuals were studied by each sample
Fig. 1 Distribution map of the sardine species included in the present study
123 1080 Eur Food Res Technol (2011) 232:1077–1086 for the methodological validation and commercial samples using the Sequence Analysis software v.5.3.1. (Applied from amounts of tissue comprising between 100 and Biosystems). 300 mg, the extraction of DNA was carried out by the The sequences were analyzed with the Chromas 1.45 CTAB method previously described, subsequent purifica- software [9] and aligned with Clustal W [10] available in tion by means of the NucleospinÒExtract II kit (Macherey– the program BioEdit 7.0 [11]. The nucleotide sequences Nagel) following the supplier’s protocol. obtained were submitted to the GeneBank database of the The quality and quantity were determined by measuring National Centre for Biotechnology Information (NCBI). of the absorbance at 260 nm and the 260/280 nm and 234/260 ratios [8] using a NanoDropTM ND-1000 spec- Development of FINS methodology trophotometer (Thermo Scientific). DNA extractions were appropriately labeled and stored at -80 °C for subsequent Phylogenetic analyses were carried out using the software tasks. Mega 4.0 using the Tamura–Nei model to calculate the genetic distances between sequences [12]. The inference of PCR amplification and DNA sequencing the phylogenetic tree was carried out with the Neighbor– Joining method [13]. The reliability of the clades formed at A fragment of the cyt b gene was amplified using the the species level in the tree was evaluated by means of a primers C-CB280F (50-TGC ATT TAC GCC CAC ATT bootstrap test with 2,000 replications. GGC CGA GG-30) and C-CB425dR (50- CCT CAG AAD GAC ATT TGB CCT CA-30), described by Jerome et al. Detection of mixture of Sardina pilchardus [4, 5]. and Sardinella aurita by means of single nucleotide In all cases, the PCR reactions were carried out in a total polymorphism analysis systems volume of 50 lL with the following composition: 100–300 ng of DNA template was added to a PCR mix Cyt b sequences obtained from Sardina pilchardus and consisting of 0.8 mM dNTP mix (Bioline), 5 lL109 Sardinella aurita were aligned with Clustal W, included in TM buffer, 2 mM MgCl2, 0.75 units of BioTaq DNA poly- BioEdit 7.0 [11] and edited to generate reliable consensus merase (Bioline), 0.8 lM of each primer, and molecular sequences for each species. For each position in the biology grade water (Eppendorf) up to adjust to the final alignment obtained, if a consensus could not be found an X volume. was used to indicate ambiguity. By comparing the con- Polymerase chain reactions were carried out in a My- sensus of each species in the alignment, nucleotide dif- CyclerTM thermocycler (BIO-RAD). Conditions of cycling ferences (SNP) were identified. were as follows: a preheating step at 94 °C for 5 min, 35 The determination of the limit of detection of the cycles of amplification (94 °C for 20 s, 56 °C for 20 s, methodology developed was established from DNA dilu- 72 °C for 20 s), and a final extension step of 72 °C for tions and mixtures of tissues of specimens belonging to 7 min. Sardina pilchardus and Sardinella aurita. In the first case, PCR amplicons were visualized on 2% agarose gels the range of extracted DNA from species varied between (Sigma) in 1X TBE buffer (Sigma) with 0.3 lg/mL of 250 and 1 ng/lL. The dilutions were prepared by adding ethidium bromide (Sigma). DNA fragments were visual- DNA from S. pilchardus to the S. aurita DNA until com- ized using the Molecular Imager Gel Doc XR System pleting the final amount. In the second case, the mixtures transilluminator and the software Quantity One_v 4.5.2 S. pilchardus/S. aurita were prepared using percentages (BIO-RAD). The 50 Base Pair Ladder (TrackltTM, Invit- from 100 to 5% of tissue. The DNA extraction was per- rogen) DNA marker was used to estimate the size of the formed from these mixtures of tissue to evaluate the min- amplicons. imum ratio S. pilchardus/S. aurita tissue that can be Next, double-stranded DNA were purified using the detected with the developed methods. NucleospinÓ 96 Extract II (Macherey–Nagel) according to the manufacturer’s instructions. The concentration and Methodological validation purity were measured by means of the Nano-DropTM ND- 1000 spectrophotometer (Thermo Scientific) as described Individuals from different species were authenticated on for DNA extraction. Subsequently, PCR products were the basis of their morphological traits. The treatment sequenced with the primers used for amplification in an applied to canned samples involved 121 °C of temperature automatic DNA Genetic Analyzer (ABI Prism 3130 and 1.2 bars of overpressure and different kinds of sauces Genetic Analyzer) using the BigDye Terminator cycle and condiments were used. The time varied depending on sequencing kit v1.1 (Applied Biosystems) following the the size of the can. These treatments were carried out in the manufacturer’s recommendations. Raw data were analyzed pilot plant of CECOPESCA (Spanish National Centre of 123 Eur Food Res Technol (2011) 232:1077–1086 1081
Fish Processing Technology). Next, products were ana- pickled sauce. It is worth highlighting this sauce because lyzed with the methodology developed in the present work. its low pH produces higher DNA degradation. The coincidence percentage between identified species on the basis of morphological traits and the genetic method- Development of FINS methodology ology developed was calculated to establish the specificity of the method. The FINS technique described by Bartlett and Davidson [21] was used in the present study to develop an identifi- Application to commercial products cation method for sardine and related species. Sequences of unknown samples are compared with sequences belonging After the validation of the methods developed in the to pattern specimens on the basis of this technique. present work, these were applied to 83 commercial samples The genetic distances between the cyt b gene sequences which includes, among others, canned products labeled as of all the studied species were estimated using the Tamura– sardine. These products were acquired in supermarkets Nei method [22]. The phylogenetic analysis of the ampli- from around the world since 2006–2010. The purpose of fied fragment (97 bp without primers) was carried out, these analyses was to evaluate the situation regarding the allowing to establishing the relationships among species by labeling of these products on the market. means of the construction of phylogeny using the data set. The phylogenetic tree contains species from two fami- lies belonging to order Clupeiformes: Cupleidae and Pri- Results and discussion stigasteridae. All the sequences belonging to individuals of the same species were grouped in the same cluster, PCR amplification allowing their identification. The bootstrap method can be used to obtain the support of the different groups obtained Drastic thermal treatments to which raw material is sub- in the phylogenetic tree. It has been calculated that boot- jected to produce the final products, degrade the DNA and strap values higher or equal to 70 usually correspond to a produce a drastic reduction in size of DNA fragment. This probability higher or equal to 95% that the corresponding is the case of canned sardines, the high temperatures and cluster is real [23], giving a quantitative measurement of pressure to which they are subjected during manufacturing, the certainty of the assignment of a sample to a particular prevent the amplification of large fragments of DNA. In species. In the phylogenetic tree of sardines herein devel- this work, obtained PCR products from amplification and oped, the large majority of clades are strongly supported sequencing of DNA with the selected primers generated an with a bootstrap value of 99 (Fig. 2). amplicon of 145 bp in all the species included in the present study (Table 1). The sequences obtained in this 97 work was submitted in NCBI database (accession numbers Etrumeus teres HQ896316–HQ896357). It is worth highlighting that for 99 Ambligaster sirm 99 Sprattus sprattus many species found that their sequences included into 96 Sardinella aurita NCBI had not been properly assigned with the correct 99 Sardinella longiceps 99 species. Sardinella maderensis Other authors have considered this problem in different 99 Sardinella albella 99 Opisthonema bulleri groups: Santaclara et al. in squids [14], Espin˜eira et al. in 75 Opisthonema medirastre scombroids, salmonids, and bivalves [15–17], Lago et al. in 99 Nematolosa vlaminghi 99 Horse Mackerels [18], and Espin˜eira et al. in a large group Herklotsichthys quadrimaculatus 98 of cephalopods [19]. But all of them establishing a maxi- Clupea harengus 99 Sardina pilchardus mum fragment size in canned products around 200 bp to 88 Sardinops sagax 75 ensure the amplification. Sardinops melanostictus 99 Ethmalosa fimbriata On the other hand, different spices and sauces used in 99 Gilchristella aestuaria 99 the manufacturing of different types of canned products Strangomera bentincki produce differences in the quantity and quality of the 99 Hyperlophus vittatus 99 Ilisha africana extracted DNA, and can decrease DNA amplification even 99 Ilisha elongata to inhibit it [17, 20]. The great majority of spices and sauces which to be found in canned sardines were validated 0.02 with the developed methodology after their manufacturing Fig. 2 Neighbor–joining tree showing the relationships among the in the pilot plant of CECOPESCA. In contrast to studies studied species, carried from the alignment of 145 bp of the cyt b published to date of this taxonomic group, was also applied gene (fragment of 97 bp without primers) 123 1082 Eur Food Res Technol (2011) 232:1077–1086
Also, the interspecific distances were calculated to know changes. The 20% of the transversions corresponding to the degree of divergence between all species included in C/G (10%) and T/A (10%) changes (Table 3). According this work (Table 2). The values for interspecific distances to Kocher et al. [24] who showed that within a particular ranged from 12.1 and 28.2%, except the distance between species, and also between closely related species, transi- Sardinops sagax and Sardinops melanostictus which was tions are more common than transversions are, transver- 1% (Table 2: bold values). This reflects the close proximity sional mutations were most frequent in the more distantly between these two species of the Sardinops genus. The related taxa. All mutational events occurred at third posi- lower value (d = 0.121) belongs to the distance between tions and were synonymous. both species of Ilisha: Ilisha africana and Ilisha elongate. The existence of the heterozygous for each SNP site This also reflects the proximity between these two species diagnostic were informative and showed mixed of species of the Ilisha genus. On the other hand, the higher value S. pilchardus/S. aurita (Fig. 3). (d = 0.282) corresponds to the distance between Clupeidae Serial dilutions of genomic DNA extracted from species family and Pristigasteridae family, specifically between of the S. pilchardus and S. aurita were tested to assess the Nematolosa vlaminghi and Ilisha africana. This distance sensitivity of the methodology developed. The total DNA reflects the separation between these two species belonging quantities used for the PCR reactions were obtained by to different families. The obtained distances for all species mixing DNA from S. pilchardus with decreasing amounts included in this work coincide with the results obtained in of DNA from S. aurita. The detection limit for detection of the phylogenetic tree. The species with lower values of mixed species, employing dilutions of genomic DNA, was genetic distances are grouped next or very close, on the 10 ng for two species. contrary, species with higher values of genetic distances Also, DNA extractions were performed from mixtures are those that are farther apart on the phylogenetic tree. The of tissues from S. pilchardus and S. aurita to estimate the proposed strategy of amplification allows the amplification optimal amount of tissue necessary to obtain a high sen- of a DNA fragment sufficiently long to discriminate suc- sitivity. Specifically DNA was extracted from 300 mg of cessfully all the sardines included in this work, even in mixtures of tissue from S. pilchardus and S. aurita in dif- canned products in which the DNA is highly degraded. ferent proportions. The diagnostic method herein designed was applied to Detection of mixture of Sardina pilchardus these mixtures, allowing the establishment of the minimum and Sardinella aurita by means of single nucleotide amount of S. aurita tissue which can be detected in the polymorphism analysis systems conditions previously described. The detection limit is lower than 5% of S. aurita tissue using 300 mg of tissue for The current situation characterized by a global market DNA extraction. makes possible to find fish from different fisheries around The results obtained in the evaluation of specificity and the world available to consumers. In order to satisfy the sensitivity of methodology developed show that this tech- high demand for sardines and sardine-type products to the nique is highly reliable. consumer, have been introduced various species with similar characteristics inside the processing industry of Methodological validation sardines. This increases the chances of finding adulterated products which contain a different species to declared The aim of the methodological validation was to check species on the label, or products which contain mixture of whether the manufacturing process which the processed different species. food were undergone, had not influence on the identifica- One of the most common substitute species to canned tion of these species. S. pilchardus is S. aurita, due to the strong resemblance of Different canned were prepared in the pilot plant of his flesh. Therefore, there is a need to develop a method- CECOPESCA simulating the conditions used in the food ology that allows not only identify the species contained in industry. This approach is useful to assess the functioning these products but also to detect mixtures of both species in and optimizing the conditions of the developed methodol- these products, in order to ensure correct labeling and ogy. The standard individuals were subjected to several defend the rights of both producers and consumers. transformation processes, allowing to evaluate the influ- From sequences obtained from the different species ence of different variables on the genetic method herein belonging to S. pilchardus and S. aurita consensus proposed. sequences were built. Twenty different single nucleotide All identified species in these samples by means of the polymorphisms highly conserved (presents in all analyzed method herein developed were in agreement with those samples) were here identified. A total of 80% of mutations based on morphological characters. Therefore, the meth- were transitions, and involved C/T (70%) and G/A (10%) odology shows a specificity of 100%. 123 u odRsTcnl(01 232:1077–1086 (2011) Technol Res Food Eur
Table 2 Genetic distances between sequences from the species included in this work Spil Sau Salb Slon Smad Smel Ssag Sspr Sben Iafr Ielo Asir Char Efim Eter Gaes Hqua Hvit Nvla Obul
Spil Saur 0.224 Salb 0.234 0.223 Slon 0.240 0.142 0.204 Smad 0.238 0.136 0.150 0.176 Smel 0.211 0.184 0.214 0.214 0.221 Ssag 0.211 0.184 0.214 0.210 0.227 0.010 Sspr 0.274 0.136 0.236 0.194 0.185 0.240 0.243 Sben 0.252 0.243 0.236 0.257 0.246 0.217 0.223 0.166 Iafr 0.234 0.252 0.223 0.197 0.234 0.252 0.252 0.233 0.184 Ielo 0.221 0.248 0.218 0.218 0.220 0.238 0.243 0.209 0.202 0.121 Asir 0.251 0.155 0.243 0.204 0.183 0.165 0.160 0.204 0.220 0.214 0.218 Char 0.211 0.186 0.244 0.221 0.196 0.188 0.193 0.163 0.169 0.249 0.191 0.205 Efim 0.244 0.165 0.233 0.181 0.173 0.233 0.238 0.165 0.168 0.204 0.189 0.184 0.176 Eter 0.222 0.148 0.223 0.200 0.183 0.177 0.177 0.206 0.208 0.225 0.218 0.144 0.209 0.157 Gaes 0.226 0.184 0.252 0.239 0.229 0.233 0.243 0.172 0.217 0.194 0.209 0.194 0.225 0.175 0.225 Hqua 0.240 0.209 0.252 0.254 0.199 0.218 0.214 0.238 0.254 0.204 0.233 0.170 0.246 0.228 0.220 0.199 Hvit 0.267 0.233 0.235 0.230 0.212 0.255 0.260 0.197 0.212 0.189 0.194 0.235 0.244 0.189 0.236 0.189 0.272 Nvla 0.223 0.214 0.233 0.230 0.214 0.223 0.228 0.194 0.217 0.282 0.238 0.243 0.212 0.243 0.216 0.252 0.194 0.269 Obul 0.272 0.217 0.162 0.219 0.166 0.172 0.178 0.230 0.221 0.214 0.180 0.220 0.195 0.230 0.250 0.236 0.251 0.200 0.236 Names of species are indicated by a capital letter representing the first letter of the genus followed by the first three letters of the species Bold values represent the highest and lowest values of genetic distance 123 1083 1084 Eur Food Res Technol (2011) 232:1077–1086
Table 3 SNP in mixture of Sardina pilchardus and Sardinella aurita In addition, only products made from fish of the species S. pilchardus S. aurita Mixed species S. pilchardus may be marketed under the commercial name of canned sardines while products made from other species SARSNP-7 T C Y are called Sardine X, having to refer to the country, geo- SARSNP-10 T C Y graphical area, species, or common name of the species in SARSNP-13 C G/A S/M accordance with the law and custom of the country where SARSNP-16 C T Y the product is sold, and always in a way that does not SARSNP-19 T C Y mislead consumers. SARSNP-25 C T Y As discussed in the review of Codex standards and SARSNP-34 A C M related texts by the Codex Alimentarius Commission in SARSNP-40 C T Y July 2001 [25], the main problem of this standard is that is SARSNP-49 T C Y somewhat confusing for consumers because under the SARSNP-61 T C Y denomination Sardine X may include various species of SARSNP-62 T C Y different genres, and from different families. This group SARSNP-64 G A R includes a large number of very similar morphologically SARSNP-73 A G R species but not all have the same commercial value and the SARSNP-76 T C Y same quality. The labeling does not provide conclusive SARSNP-82 T C Y information to consumers about product quality, so in some SARSNP-85 T A W countries expressed that should be avoided to use a com- SARSNP-88 T C Y mercial denomination which includes a same name (Sar- SARSNP-91 T C Y dine) to designate products so different. In addition, this SARSNP-95 T C Y represents a economical loss for exporting countries of SARSNP-97 A T W S. pilchardus because of unfair competition exerted by similar species, that the standard designates as sardines X Y = C ? T; M = A ? C; W = A ? T; R = G ? A and which by allusion intended benefit of a commercial tradition and secured quality. In this sense, one of the main objectives of this meth- odology is that by means of this technique can remove any doubts that may arise with the ambiguity of the name sardines X. The developed methodology was applied to 83 com- mercial samples labeled as sardine. This application allowed knowing the degree of fulfillment of the labeling regulations in these products. All commercial samples tested were identified as S. pil- chardus except 13 of them (15.66%), all canned. Of these, 10 labeled samples as canned sardine contained S. aurita instead of S. pilchardus, and 2 labeled samples as canned Fig. 3 SNP chromatogram in mixture of Sardina pilchardus and sardine contained S. longiceps instead of S. pilchardus.In Sardinella aurita. Y = C ? T; S = C ? G; M = C ? A; addition, 1 labeled sample as canned sardine contained R = G ? A; W = T ? A. SNPs positions. Y: 7, 10, 16, 19, 25, 40, mixture of S. aurita and S. pilchardus. 49, 61, 62, 76, 82, 88, 91 and 95; S: 13; M: 13 and 34; R: 64 and 73; W: 85 and 97 The results showed that the most common species used as substitute species in canned sardines are S. aurita fol- Application to commercial products lowed by S. longiceps.
The Codex Stan 94 provides that canned sardines or sar- dine-type products are prepared with fresh or frozen fish Conclusions from a list of 21 species, enphasizing S. pilchardus.This standard applies to canned sardines and sardine-type In the present work, one method based in the DNA analysis products in water, oil or other suitable sauces, and does not that allow the genetic identification of the most important apply to another special products, where the content of commercialized species of sardine and related species was these fishes are less than 50% of net content of the canned. been developed.
123 Eur Food Res Technol (2011) 232:1077–1086 1085
As sardine-type products also include anchovies [3]. As 4. Jerome M, Lemaire C, Bautista JM, Fleurence J, Etienne M can see, have not been included species of the genus (2003) Molecular phylogeny and species identification of sar- dines. J Agric Food Chem 51:43–50 Engraulis in this work. This is because their main form of 5. Jerome M, Lemaire C, Verrez-Bagnis W, Etienne M (2003) marketing is as semi-canned. In this case, the DNA is less Direct sequencing method for species identification of canned degraded than in the case of canned products. Therefore, it sardine and sardine-type products. J Agric Food Chem is possible to use the methodology developed by Santaclara 51:7326–7332 6. Whitehead PJP (1985) FAO species catalogue. Vol 7. Clupeoid et al. [26] in these cases, allowing the identification of all fishes of the world (suborder Clupeioidei). An annotated and species of the genus Engraulis. illustrated catalogue of the herrings, sardines, pilchards, sprats, The main advantage of this method is that besides shads, anchovies and wolf-herrings. Part 1-chirocentridae, clu- including a high number of species (21 species), it is based peidae and pristigasteridae. FAO Fish Synop 125(7/1):1–303 7. Roger SO, Bendich AJ (1988) Extraction of DNA from plant on the use of a unique pair of primers which allows the tissues. Plant Mol Biol Man A6:1–10 amplification of a fragment with a size of 145 bp in all 8. Winfrey MR, Rott MA, Wortman AT (1997) UnraVeling DNA: studied species of sardines, and that can be applied to all molecular biology for the laboratory. Prentice Hall, New York kinds of processed products with different spices and 9. Mc Carthy C (1996) Chromas version 1.45. School of Health science, Griffifth University, Gold Coast Campus, Queensland, sauces, included these that have been undergone intensive Australia transformation processes. Adulteration or possible food 10. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins frauds are a financial problem that affects many products DG (1997) The CLUSTAL_X windows interface: flexible strat- such as canned sardines, where the declared product is egies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 often substituted or adulterated by species with lower 11. Hall TA (1999) BioEdit: a user-friendly biological sequence commercial value. Thus, it is noteworthy that this meth- alignment editor and analysis program for Windows 95/98/NT. odology allows to detect the presence of mixtures of Nucl Acids Symp Ser 41:95–98 S. pilchardus and S. aurita in the final product with a limit 12. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. of detection of 5% of tissue (w/w). Mol Biol Evol 24:1596–1599 The importance of this work is that allows to identify 13. Saitou N, Nei M (1987) The neighbor-joining method—a new most species of sardines and their possible applications are method for reconstructing phylogenetic trees. Mol Biol Evol the following: normative control of raw and processed 4:406–425 14. Santaclara FJ, Espin˜eira M, Vieites JM (2007) Genetic identifi- products, particularly the authenticity of imported species, cation of squids (families Ommastrephidae and Loliginidae) by verification of the traceability of different fishing batches PCR-RFLP and FINS methodologies. J Agric Food Chem along the commercial chain, correct labeling, protection of 55:9913–9920 the consumer’s rights, fair competence among fishing 15. Espin˜eira M, Gonzalez-Lavı´n N, Vieites JM, Santaclara FJ (2009) Development of a method for the identification of scombroid and operators, and fisheries’ control. common substitute species in seafood products by FINS. Food Chem 117:698–704 Acknowledgments We also thank Hideo Sakai (National Research 16. Espin˜eira M, Gonzalez-Lavı´n N, Vieites JM, Santaclara FJ (2009) Institute of Fisheries Science. Tosa Bay, Japan), Jose A. Gonza´lez Development of a method for the genetic identification of com- (ICCM, Spain), Sean Fennessy (Oceanographic Research Institute, mercial bivalve species based on mitochondrial 18S rRNA South Africa), Kim Smith and Daniel Gaughan (Estuarine & Inshore sequences. J Agric Food Chem 57:495–502 Fisheries Australia), Victor Otecio (Universidad Laica Eloy Alfarode 17. Espin˜eira M, Vieites JM, Santaclara FJ (2009) Development of a Marabi), Aland Connell (South Africa), Konstantinos Triantafyllidis genetic method for the identification of salmon, trout, and bream (University of Thessaloniki, Greece), Jorge Castillo Pizarro (Instituto in seafood products by means of PCR–RFLP and FINS meth- de Fomento Pesquero, Chile), Dianne J Bray (Museum Victoria, odologies. Eur Food Res Technol 229:785–793 Australia), Grigorius Krey (Fisheries Research Institute, Greece), 18. Lago FC, Herrero B, Vieites JM, Espin˜eira M (2011) Genetic Technical University of Denmark for kindly supplying the sardines identification of horse mackerel and related species in seafood species samples. This work was funded by the European Fisheries products by means FINS methodology. J Agric Food Chem Fund (EFF) and the Ministerio de Madio Ambiente y Medio Rural y 59:2223–2228 Marino (Secretarı´a General del Mar) under the order ARM/1193/2009 19. Espineira M, Vieites JM, Santaclara FJ (2010) Species authenti- of the 6th of May. cation of octopus, cuttlefish, bobtail and bottle squids (families Octopodidae, Sepiidae and Sepiolidae) by FINS methodology in seafoods. Food Chem 121:527–532 References 20. Ram JL, Ram ML, Baidoun FF (1996) Authentication of canned tuna and bonito by sequence and restriction site analysis of 1. Margalef R, Andreu B (1958) Componente vertical de los polymerase chain reaction products of mitochondrial DNA. movimientos del agua de la rı´a de Vigo y su posible relacio´n con J Agric Food Chem 44:2460–2467 la entrada de la sardina. Investigacio´n Pesquera 11:105–126 21. Bartlett SE, Davidson WS (1992) FINS (forensically informative 2. Miranda A, Cala RM, Iglesias J (1990) Effect of temperature on nucleotide sequencing): a procedure for Identifying the animal the development of eggs and larvae of sardine Sardina pilchardus origin of biological specimens. Biotechniques 12:408–411 Walbaum in captivity. 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23. Hillis DM, Bull JJ (1993) An empirical-test of bootstrapping as a 25. Codex Alimentarius Commission (2001) ALINORM 01/21. Vol method for assessing confidence in phylogenetic analysis. Syst I—Add. 2. Joint FAO/Who food standards programme. Consid- Biol 42:182–192 eration of standards and related texts at step 8 or equivalent 24. Kocher TD, Thomas WK, Meyer A, Edwards SV, Paabo S, 26. Santaclara FJ, Cabado AG, Vieites JM (2006) Development of a Villablanca FX, Wilson AC (1989) Dynamics of mitochondrial method for genetic identification of four species of anchovies: E. DNA evolution in animals: amplification and sequencing with encrasicolus, E. anchoita, E. ringens and E. japonicus. Eur Food conserved primers. Proc Natl Acad Sci USA 86:6196–6200 Res Technol 223:609–614
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Development of a FINS- based method for the identification of skates species of commercial
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Food Control 24 (2012) 38e43
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Food Control
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Development of a FINS- based method for the identification of skates species of commercial interest
Fátima C. Lago, Juan M. Vieites, Montserrat Espiñeira*
Area of Molecular Biology and Biotechnology, ANFACO-CECOPESCA, Vigo, 36310 Pontevedra, Spain article info abstract
Article history: Rajidae family is one of the most diverse families within Batoidea superorder with more of 220 described Received 3 June 2011 species, of which many skates are particularly vulnerable to overfishing. This fact makes IUCN Received in revised form (International Union for Conservation of Nature) Red List of Threatened Species contains 209 of these 24 August 2011 skate species. Accepted 25 August 2011 The main marketing formats of these species are fresh and frozen wings. This makes impossible their identification on the basis of their morphological characters. For these reasons, in the present study Keywords: a method for genetic identification of different skate species has been developed. The technique is based Skate fi Ray on sequencing of a fragment of 555 bp from ampli ed DNA, COI (Cytochrome Oxidase subunit 1) gene, by Rajidae PCR and subsequent phylogenetic analysis (FINS: Forensically Informative Nucleotide Sequencing). The Genetic identification technique allows the genetic identification of more than 40 skate species in skate products. FINS The main novelty of this work lies in the fact that up to now there is no work about the genetic PCR identification that includes so many skate species. Therefore, this molecular tool is appropriate to clarify questions related with the correct labeling of skate commercial products, the traceability of raw materials, and the control of imported skates, and also can be applied to questions linked to the control of skate fisheries. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Species, 3 are in critical condition, 7 endangered, 13 are vulnerable and the rest of them are in less of a threat (IUCN, 2011). In Europe Skates and Rays belong to Batoidea superorder which contain- dramatic population declines have taken place in some skate which ing more than 500 described species in thirteen families. Into this recently are listed as ‘Critically Endangered’ on the IUCN Red List of superorder, the Rajidae is one of the most diverse families with Threatened Species. An example of endangered skate species is more of 220 described species. They are found worldwide in marine represented by the disappearance of Rostroraja alba in the Irish Sea waters, from the Atlantic to Pacific Oceans and are closely related to (Chevolot, 2006; Dulvy et al., 2000). sharks, from which can be distinguished by their flattened bodies, The total world catch of skates in recent years was approxi- enlarged pectoral fins fused to their head, and gill slits placed on mately 250,000 tons (Seafish, 2009). These data show their great their ventral surfaces. Strictly speaking, species from this family are consume demand of this group of species and their importance as referred as skates, whereas species belonging to the Myliobatidae a resource for human consumption. To avoid the excessive exploi- family are rays. On the other hand, in European waters, short-nosed tation of these resources in the future as a result of the catch rates, rajid species are colloquially referred to as rays and long-nosed and also the disappearance of skate species whose stocks are in species as skates (Dulvy, Metcalfe, Glanville, Pawson, & Reynolds, continuous decline, the European Union (EU) has established total 2000). Today, colloquially use the terms interchangeably in allowable catches (TAC) for skates in most European waters. These formal or informal designation of these species. In this work we will put a limit of 30,371 tons in the total quantity of skates that can be use from now on the skate term to refer to these species. catched in 2011 (Fishing TACs and Quotas, 2011) and has prohibited Many skates are particularly vulnerable to overfishing because catches for certain fish stocks of skates by the Council Regulation of their large size, slow growth, late maturity and low fecundity. No 57/2011 (Council Regulation (EU) No 57/2011). Also, European From the 209 skate species of the IUCN Red List of Threatened Commission Regulation No 43/2009 establishes regulations relative to record of landings of these species in different fishing areas * Corresponding author. Tel.: þ(34) 986 469 301; fax: þ(34) 986 469 269. (Council Regulation (EU) No 43/2009). Concerning the labeling of E-mail address: [email protected] (M. Espiñeira). skate products, the Council Regulation No 57/2011 (Council
0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.08.034 F.C. Lago et al. / Food Control 24 (2012) 38e43 39
Regulation (EU) No 57/2011) also establishes the correspondence of Table 1 latin names and common names to main commercial skate species. Species included in the present work. Skates are valued for the high quality of their meat, and the main Family Scientific name Common name Samplesa NCBI edible parts are their wings. The species identification is possible in Myliobatidae Aetobatus narinari Eagle ray 2 FJ812203 whole individuals based on morphological characteristics but it is Pteromylaeus bovinus Duckbill ray 3 impossible in processed products such as skate wings. In this sense Amblyraja badia Broad skate 2 DQ385444 there is a need to develop techniques to allow distinguishing Amblyraja radiata Thorny skate 2 AF106038 Bathyraja abyssicola Deepsea skate 3 DQ104889 different skate species, not only to protect populations which are Bathyraja aleutica Aleutian skate 5 DQ104895 endangered, but also because they all have different commercial Bathyraja brachyurops Broadnose skate 3 EU074334 values. Bathyraja eatonii Eaton’s skate 2 EU119812 Most of the molecular studies till now are focused on systematic Bathyraja interrupta Bering skate 4 Bathyraja irrasa Kerguelen 2 EU119815 of skates where several factors have been studied to estimate the sandpaper skate maturation and reproductive patterns in elasmobranch species Bathyraja lindbergi Commander 4 DQ104909 (Alvarado Bremer et al., 2005; Griffiths et al., 2010; Smith et al., skate 2008; Spies, Stevenson, Orr, & Hoff, 2011; Tinti et al., 2003; Turan, Bathyraja maculata Whiteblotched 2 DQ104911 2008; Valsecchi et al., 2005). All of them include a low number of skate Bathyraja mariposa Butterfly skate 4 DQ104916 skate species. Concerning the genetic studies of skate species, it is Bathyraja minispinosa Smallthorn skate 4 DQ104919 worth highlighting the works of Spies et al. (Spies, Gaichas, Bathyraja multispinis Multispine skate 2 Stevenson, Orr, & Canino, 2006) and Smith et al. (Smith et al., Bathyraja murrayi Murray’s skate 4 EU119844 2008), who studies COI and Cyt b fragments in skate species. In Bathyraja parmifera Alaska skate 7 DQ104920 Bathyraja trachura Black skate 2 DQ104933 both cases the number of species included is low. Bathyraja violacea Okhotsk skate 2 FJ164395 In this study, genetic authentication of more of 40 species of Cruriraja Roughnose 2 skates living around the world have been developed by means of parcomaculata legskate FINS analysis of a partial sequence of the mitochondrial DNA Dipturus australis Sydney skate 2 DQ108188 (mtDNA) COI gene. This methodology has previously been used Dipturus cerva White-spotted 2 DQ108189 fi skate successfully to identify different species such as angler sh species Rajidae Dipturus innominata Smooth skate 2 (Espiñeira, Gonzalez-Lavin, Vieites, & Santaclara, 2008), scombroid Dipturus nidarosiensis Norwegian 2 species (Espiñeira, Atanassova, Vieites, & Santaclara, 2010), skate salmonid species (Espiñeira et al., 2010), cephalopod species Dipturus oxyrinchus Long-nosed 2 skate (Espiñeira et al., 2010; Santaclara, Espineira, & Vieites, 2007), Dipturus Slime skate 5 pelagic species (Lago, Herrero, Madriñán, Vieites, & Espiñeira, pullopunctatus 2011), and meat products too (Lago et al., 2011). Dipturus springeri Roughbelly 2 The particularity of this work lies in the fact that up to now there skate is no work about the genetic identification that includes so many Dipturus whitleyi Wedgenose 2 DQ108190 skate species. Taking into account the existence of a global market, the Leucoraja naevus Sandy ray 2 need arises for developed identification techniques that take into Raja asterias Starry skate 2 account a maximum number of species which are susceptible to be Raja binoculata Big skate 7 GU440489 marketed under the commercial denomination of skate, and allows Raja clavata Thornback ray 2 HM043196 Raja inornata California skate 2 GU440490 to improve the quality of fish products by means of the guarantee Raja miraletus Brown ray 3 that offers a correct traceability and authenticity. Raja montagui Spotted ray 2 Raja rhina Longnose skate 5 DQ104882 2. Materials and methods Raja stellulata Starry skate 2 GU440492 Raja straeleni Biscuit skate 2 Rajella dissimilis Ghost skate 2 2.1. Sample collection and storage Rhinoraja taranetzi Mud skate 5 DQ104930 Rostroraja alba Bottlenose skate 3 Samples of different skates (between 3 and 10 individuals by Zearaja nasuta Brown rough 2 each sample) have been collected from several locations around the skate world (Table 1 and Fig. 1). Whenever possible, the individuals have a Between 3 and 10 individues were studied by each sample. been identified according to morphological characters (Compagno, 1984a, 1984b; Serena, 2005). In other cases, ethanol preserved fish tissues have been provided by Universities and research centers located around the world. Once identified, samples have been The quality and quantity were determined by measuring of the labeled and preserved at 80 C. absorbance at 260 nm and the 260/280 nm and 234/260 ratios Moreover, 20 products labeled as Skate or Ray have been (Winfrey, Rott, & Wortman, 1997) using a NanoDropÔ ND-1000 provided by import industries or purchased in supermarkets and spectrophotometer (Thermo Scientific). DNA extractions were shops from Spain, in order to apply the developed methodology to appropriately labeled and stored at 80 C for subsequent tasks. commercial samples (Table 2).
2.2. DNA extraction 2.3. PCR amplification and DNA sequencing
Genomic DNA has been extracted from 30 mg of muscle in fresh COI gene sequences of different skate species have been and frozen samples, according to the method described by Roger downloaded from the National Center for Biotechnology Informa- and Bendich with slight modifications (Roger & Bendich, 1988). The tion (NCBI) (Table 1). These have been aligned with BioEdit 7.0 obtained DNA has been diluted in 100 mL of 1X Trise EDTA (TE) (Hall, 1999) and for them, a primer set has been designed by hand. buffer (Sigma). The name and sequence of the forward and reverse primers are, 40 F.C. Lago et al. / Food Control 24 (2012) 38e43
Fig. 1. Distribution map of skates included in the present study.
respectively, COI_RajaF, 50-CCG CTT AAC TCT CAG CCA TC-30, and Polymerase chain reactions have been carried out in COI_RajaR, 50-TCA GGG TGA CCA AAG AAT CA-30, and the length of a MyCyclerÔ thermocycler (BIO-RAD). Conditions of cycling have the amplicon produced by these primers is 555 bp (Fig. 2). been as follows: a preheating step have been an initial 3 min pre- In all cases the Polymerase Chain Reactions (PCR) have been heating step at 95 C, followed by 35 cycles (30 s at 95 C, 30 s at carried out in a total volume of 50 mL with the following compo- 50 C, and 30 s at 72 C), and a final extension step of 3 min at 72 C. sition: 50e100 ng of DNA template have been added to a PCR mix consisting of 0.8 mM dNTP mix (Bioline), 5 mL 10X buffer, 2 mM MgCl2, 0.75 units of BioTaqÔ DNA polymerase (Bioline), 0.8 mMof each primer and molecular biology grade water (Eppendorf) up to adjust to the final volume.
Table 2 Commercial samples analyzed with the method developed.
Sample Codea Products Species labeled Species identifiedb S1 Fresh skate whole Raja spp. Amblyraja radiata S2 Raja spp. Bathyraja brachyurops S3 Raja spp. Leucoraja naevus S4 Raja spp. Raja clavata S5 Raja spp. Raja miraletus S6 Fresh skate wings Raja spp. Amblyraja radiata S7 Raja spp. Bathyraja brachyurops S8 Raja spp. Bathyraja brachyurops S9 Raja spp. Leucoraja naevus S10 Raja spp. Raja clavata S11 Frozen skate wings Raja spp. Amblyraja radiata S12 Raja spp. Amblyraja radiata S13 Raja spp. Amblyraja radiata S14 Raja spp. Bathyraja brachyurops S15 Raja spp. Bathyraja brachyurops S16 Raja spp. Bathyraja brachyurops S17 Raja spp. Bathyraja brachyurops S18 Raja clavata Bathyraja multispinis S19 Raja spp. Leucoraja naevus S20 Raja spp. Raja clavata
a Code shown in Fig. 3 that locates the commercial samples in the phylogenetic tree of the studied species. Fig. 2. PCR products of fresh and frozen samples obtained in the PCR (Size obtained: b Species in bold correspond with the mislabeled species. 555 bp). F.C. Lago et al. / Food Control 24 (2012) 38e43 41
PCR amplicons have been visualized on 2% agarose gels (Sigma) and rapid rate of evolutionary change of mtDNA compared to in 1X TBE buffer (Sigma) with 0.3 mg/mL of ethidium bromide nuclear DNA to have make it a suitable tool for genetic studies of (Sigma). DNA fragments have been visualized using the Molecular several fish groups at multiple taxonomic levels (Espiñeira et al., Imager Gel Doc XR System transilluminator and the software 2010; Espiñeira et al., 2008; Lago et al., 2011; Santaclara, Cabado, Quantity One_ v 4.5.2 (BIO-RAD). The 100 Base Pair Ladder & Vieites, 2006; Santaclara et al., 2007) The COI sequence is one (TrackltÔ, Invitrogen) DNA marker has been used to estimate the of the mtDNA gene which has been widely used as molecular size of the amplicons. markers in the genetic identification of a great number of species Next, double-stranded DNA have been purified using the belonging to different taxa and that widely adopted to discriminate Ó Nucleospin 96 Extract II (MachereyeNagel) according to the among related species of marine organisms (Alvarado Bremer et al., manufacturer’s instructions. The concentration and purity have 2005; Espiñeira et al., 2008; Smith et al., 2008; Spies et al., 2006, been measured by means of the NanoDropÔ ND-1000 spectro- 2011). Concerning the genetic studies of skate species, it is worth photometer (Thermo Scientific) as described for DNA extraction. highlighting the work of Spies et al. (Spies et al., 2006), who Subsequently, PCR products have been sequenced with the primers identified 15 skate species by means of amplification and subse- used for amplification in an automatic DNA Genetic Analyzer (ABI quent sequencing of a COI fragment and found shallow divergences Prism 3130 Genetic Analyzer) using the BigDye Terminator cycle among 13 of 15 species. Also noteworthy is the work of Smith et al. sequencing kit v1.1 (Applied Biosystems) following the manufac- (Smith et al., 2008), who studies Cyt b and COI fragments in 9 skate turer’s recommendations. Raw data have been analyzed using the species by amplification and sequencing and obtains evidences for Sequence Analysis software v.5.3.1. (Applied Biosystems). a new species. In both cases the number of species included is low. The sequences have been analyzed with the Chromas 1.45 In this work, the COI gene has been evaluated to identify all of software (Mc Carthy, 1996) and aligned with Clustal W (Thompson, the species herein contained. Amplification and sequencing of PCR Gibson, Plewniak, Jeanmougin, & Higgins, 1997) available in the products DNA amplificated with the primers COI_RajaF and program BioEdit 7.0 (Hall, 1999). The nucleotide sequences ob- COI_RajaR have generated an amplicon of 555 bp (515 bp without tained have been submitted to the GeneBank database of the primers) in all the species included in the present study (accession National Center for Biotechnology Information (NCBI). numbers JN602376-JN602438)(Fig. 2).
2.4. Development of FINS methodology 3.2. FINS methodology
Phylogenetic analyses have been carried out using the software The FINS technique described by Bartlett and Davidson (Bartlett Mega 4.0 using the TamuraeNei model to calculate the genetic & Davidson, 1992) has been used in the present study to develop an distances between sequences (Tamura, Dudley, Nei, & Kumar, identification method for skate species. The basis of this technique 2007). The inference of the phylogenetic tree has been carried is the comparison of sequences of unknown samples with out with the Neighbor-Joining method (Saitou & Nei, 1987). The sequences belonging to pattern specimens. reliability of the clades formed at the species level in the tree has The genetic distances between the COI gene sequences of all been evaluated by means of a bootstrap test with 2000 replications. studied species have been estimated using the TamuraeNei method (Tamura & Nei, 1993). 2.5. Methodological validation The phylogenetic analysis of the amplified fragment (515 bp without primers) has been carried out, allowing establishing the From individuals from different skate species which have been relationships among species by means of the construction of authenticated on the basis of their morphological traits, a meth- phylogeny using the data set (Fig. 3). The phylogenetic tree ob- odological validation has been carried out. For this, the main tained shows a major node that includes all the species of the treatments applied to those commercial products, fresh and frozen Rajidae family while the species of the Miliobatidae family are treatments of the wings, have been applied to them. The fish was clearly differentiated into two independent branches. Within the frozen to 20 C in less than 2 months in freezing chambers like Rajidae family 3 great and important skate genera as are Dipturus, those used in industrial fisheries. These treatments have been Raja and Bathyraja genus can clearly be differentiate. The position carried out in the pilot plant of CECOPESCA (Spanish National of Dipturus pullopunctatus within Raja genus is to be highlighted. In Center of Fish Processing Technology). Next, products have been the great majority of the works about D. pullopunctatus that exist, analyzed with the methodology developed in the present work. such as the Walmsley-Hart’ study (Walmsley-Hart, Saber, & Buxton, The coincidence percentage between identified species by means of 1999), this species is studied as if it were a species of the Raja genus. the basis of morphological traits and by the genetic methodology It should be mentioned too, the great morphological similarities developed has been calculated to establish the specificity of the between this species and the others skate species of different method. genera. The genetic distance analysis shows that the most closely related species to D. pullopunctatus are Dipturus australis, Dipturus 2.6. Application to commercial products cerva and Dipturus innominata within the Dipturus genus with a genetic proximity of 0.06, but also other species of different After the validation of the methods developed in the present genera such as Zearaja nasuta with a less genetic proximity of 0.058 work, they have been applied to 20 commercial samples labeled as (data not shown). skate or rays (Table 2). These products have been acquired in On the other hand, it is also important to note that all sequences supermarkets from Spain. belonging to individuals of the same species have been grouped into the same clade allowing their identification, except Raja asterias and 3. Results and discussion Raja montagui are grouped into the same clade. This indicates that this fragment of 555 bp (515 bp without primers) of COI gene does 3.1. PCR amplification not allow differentiating between these two species of the genus Raja. These clades are supported by bootstrap values greater than MtDNA analysis is a useful molecular marker for systematics 97, except for Bathyraja interrupta and Bathyraja mariposa which are because of its special features. The pattern of maternal inheritance values of 88 and 89 respectively. These bootstrap values reflect the 42 F.C. Lago et al. / Food Control 24 (2012) 38e43
Fig. 3. Neighbor-Joining tree obtained by the alignment of 42 partial COI gene sequences of 515 bp.
robustness of the nodes obtained and indicate a higher confidence 3.4. Application to commercial products in the correct identification of all of these species. The developed methodology has been applied to 20 commercial 3.3. Methodological validation samples labeled as skate or rays (Table 2). This application permits knowing the degree of fulfillment of the labeling regulations in The aim of the methodological validation has been to check these products. whether the manufacturing process which the processed food have Rajidae family includes a large number of very similar undergone, had not influence on the identification of these species. morphologically species but not all have the same commercial Wings of different skate species have been prepared in the pilot value. One of the major problems related to skate products is that plant of CECOPESCA simulating the conditions of freezing used in often they are sold in bulk. Due to this, this marketing kind very the food industry. This approach is useful to assess the functioning common in supermarkets involves that proper labeling of bagging and optimizing the conditions of the developed methodology. The products is not available. These products are marked with labels standard individuals have been subjected to several transformation which indicate the taxonomic group, the catchment area and in processes, allowing the evaluation and the influence of different many cases the species. It is worth highlighting that all of tested variables on the genetic method herein proposed. skate products were labeled such as Raja spp., except one which is All identified species in these samples by means of the method labeled as Raja clavata. Obtained results showed that the most herein developed have been in agreement with those based on common species used in skate products are species of Bathyraja morphological characters, except in the case of R. asterias and genus, specially Bathyraja brachyurops (35%) followed to Amblyraja R. montagui which can not be differentiated genetically. Therefore, radiata (25%) and others less common species such as Bathyraja, the methodology shows a very high specificity. Raja and Leucoraja. F.C. Lago et al. / Food Control 24 (2012) 38e43 43
4. Conclusions Fishing TACs and Quotas 2011. Griffiths, A. M., Sims, D. W., Cotterell, S. J., El Nagar, A., Ellis, J. R., Lynghammar, A., et al. (2010). Molecular markers reveal spatially-segregated cryptic species in In the present work, one method based on the DNA analysis that a critically endangered fish, the common skate Dipturus batis. Proceedings of the allows the genetic identification of the most important commer- Royal Society B, 277,1497e1503. cialized species of skates has been developed. Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and fi analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, The importance of this work is in that it allows the identi cation 95e98. of most species of skates, and their possible applications are the IUCN. (2011a). Red list of threatened species. Version 2010.4. following: normative control of raw and processed products, Lago, F. C., Herrero, B., Madriñán, M., Vieites, J. M., & Espiñeira, M. (2011). fi Authentication of species in meat products by genetic techniques. European particularly the authenticity of imported species, veri cation of the Food Research and Technology, 232, 509e515. traceability of different fishing batches along the commercial chain, Mc Carthy, C. (1996). Chromas version 1.45. Queensland, Australia: School of Health correct labeling, protection of the consumer’s rights, fair compe- Science, Griffifth University, Gold Coast Campus. fi fi ’ Roger, S. O., & Bendich, A. J. (1988). Extraction of DNA from plant tissues. Plant tence among shing operators, and sheries control. Molecular Biology Manual, A6,1e10. Saitou, N., & Nei, M. (1987). The neighbor-joining method e a new method for Acknowledgment reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4), 406e425. Santaclara, F. J., Cabado, A. G., & Vieites, J. M. (2006). Development of a method for We also thank to Allan Connell (South Africa. 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(2003). opportunities for certain fish stocks and groups of fish stocks, applicable in EU Development of molecular and morphological markers to improve species- waters and, for EU vessels, in certain non-EU waters. specific monitoring and systematics of Northeast Atlantic and Mediterranean Chevolot, M. S. O. M. (2006). Assessing genetic structure of thornback ray, Raja skates (Rajiformes). Journal of Experimental Marine Biology and Ecology, 288, clavata: A thorny situation? University of Groningen. 149e165. Dulvy, N. K., Metcalfe, J. D., Glanville, J., Pawson, M. G., & Reynolds, J. D. (2000). Turan, C. (2008). Molecular systematic analyses of Mediterranean skates (Raji- Fishery stability, local extinctions, and shifts in community in skates. Conser- formes). Turkish Journal of Zoology, 32(4), 437e442. vation Biology, 14(1), 283e293. Valsecchi, E., Pasolini, P., Bertozzi, M., Garoia, F., Ungaro, N., Vacchi, M., et al. (2005). Espiñeira, M., Atanassova, M., Vieites, J. M., & Santaclara, F. J. (2010). Validation of Rapid Miocene-Pliocene dispersal and evolution of Mediterranean rajid fauna a method for the detection of five species, serogroups, biotypes and virulence as inferred by mitochondrial gene variation. Journal of Evolutionary Biology, 18, factors of vibrio by multiplex PCR in fish and seafood. Food Microbiology, 27(1), 436e446. 122e131. Walmsley-Hart, S. A., Saber, W. H. H., & Buxton, C. D. (1999). The biology of Espiñeira, M., Gonzalez-Lavin, N., Vieites, J. M., & Santaclara, F. J. (2008). Authen- the skates Raja Wallacei and R. pullopunctata (Batoidea: Rajidae) on the tication of anglerfish species (Lophius spp) by means of polymerase chain Agulhas bank, South Africa. South African Journal of Marine Science, 21, reaction-restriction fragment length polymorphism (PCR-RFLP) and forensically 165e179. informative nucleotide sequencing (FINS) methodologies. Journal of Agricultural Winfrey, M. R., Rott, M. A., & Wortman, A. T. (1997). Unraveling DNA: molecular and Food Chemistry, 56(22), 10594e10599. biology for the laboratory. New York: Prentice Hall.
Authentication of the most important species of freshwater eels by means
of FINS
Página 91 de 153
Eur Food Res Technol (2012) 234:689–694 DOI 10.1007/s00217-012-1672-4
ORIGINAL PAPER
Authentication of the most important species of freshwater eels by means of FINS
Fátima C. Lago · Juan M. Vieites · Montserrat Espiñeira
Received: 2 November 2011 / Revised: 12 January 2012 / Accepted: 16 January 2012 / Published online: 7 February 2012 © Springer-Verlag 2012
Abstract Eels are a taxonomic group with great commer- they spend part of their life in freshwater rivers, lakes, or cial importance due to their huge steaks. They have very estuaries and return to the ocean to spawn. Eels are charac- high demand, especially the young ones. There are high terized by snake-shaped bodies and large and pointed morphological similarity and diVerent market values heads. Their dorsal Wns are usually continuous with their between diVerent species. For these reasons arises the need caudal and anal Wns, and moreover, they have small pecto- to develop techniques that allow identifying as many spe- ral Wns. Their scales are thin and soft [1]. cies as possible. In this study, a DNA method based on Eels are an important resource in the worldwide Wsher- DNA phylogenetic analysis of sequences (forensically ies. In aquaculture, both juveniles and adults are harvested, informative nucleotide sequencing) has been developed. but nowadays, these cultures are entirely dependent on nat- This method has been used to authenticate 12 eel species, ural environment catches. Eel aquaculture production is including the most important to commercial level (Anguilla based on the fattening of juvenile eels captured in the wild, anguilla, A. rostrata, A. japonica, A. australis), by means as so far has not been possible complete the life cycle of of the ampliWcation of a 239-base pair (bp) fragment of the eels in captivity [2]. mitochondrial Cytochrome b (cyt b) gene. This method is The eels are a traditional food in the Spanish Mediterra- useful to clarify questions related to the correct labeling of nean region, and they are also consumed in other European commercial products and to verify the traceability in com- countries such as Germany, Holland, Denmark, and Italy. mercial trade and for Wsheries control. However, the main consumers are in Asian countries, espe- cially in Japan, where it is considered a delicacy. They are Keywords Eels · Anguilla · PCR · IdentiWcation · primarily marketed alive but also marketed fresh or chilled, Authentication · FINS frozen, smoked and canned. Of the existing 19 species, 4 are the most consumed species: Anguilla anguilla, A. japonica, A. rostrata, and A. australis. Species such Introduction as A. marmorata, A. dieVenbachii, A. celebesensis, A. bicolor, A. nebulosa, among others are grown in aqua- Eels are teleost Wsh belonging to Anguillidae family, which culture. However, processed products from these species includes a single genus, Anguilla, commonly known as are similar to each other in external appearance and taste. freshwater eels and are distributed throughout the most Since the mid-1960s, there is a serious decline in eel tropical and temperate waters except the eastern PaciWc and catches throughout its range [3–5], which is attributed to south Atlantic. Eels are catadromous, which means that various causes such as overWshing and destruction or alteration of natural habitats. Recent Wsheries data indicate a dramatic decline in the abundance of A. anguilla, A. rostrata, and A. japonica since 1980 [2, 3, 6]. The FAO & F. C. Lago · J. M. Vieites · M. Espiñeira ( ) global catch landings of A. anguilla show that in 2005, only Area of Molecular Biology and Biotechnology, ANFACO-CECOPESCA, 36310 Vigo, Pontevedra, Spain 4,855 tonnes was caught, a decline of 76% since a harvest e-mail: [email protected] peak in 1968 [3]; for this reason, this species has been 123 690 Eur Food Res Technol (2012) 234:689–694 included in the IUCN (International Union for Conserva- Materials and Methods tion of Nature) Red List of Threatened Species. Therefore, it is necessary to take measures to protect this species. The Sample collection and storage European Community has published a regulation (EC Reg- ulation No. 1100/2007) that requires member states the Authentic specimens of diVerent eel species were obtained development of management plans for the European eel, by ANFACO-CECOPESCA (National Association of Fish reduction of at least 50% in commercial and recreational and Seafood canning Manufactures-Technical Centre for Wshing, and restocking. These regulations also require that the preservation of Fish and Aquaculture products) from July 31, 2013, 60% of eels less than 12 cm in length (between three and seven individuals of each species) from caught annually are set aside for reforestation and aquacul- several locations around the world (Fig. 1). When possible, ture [7]. the individuals were identiWed according to morphological Morphological characters change from one develop- character [1]. In other cases, ethanol-preserved Wsh tissues ment stage to another. For this reason, even if the eels are provided by universities and research centers around the marketed alive and unprocessed, identiWcation based on world were labeled and preserved at ¡80 °C. morphological traits is impossible. Molecular techniques have emerged as a crucial, eYcient, and reliable species DNA extraction identiWcation tool in the identiWcation of eels . This identi- Wcation is critical for conservation and aquaculture man- DNA extraction was performed using 30 mg of muscle tis- agement of eels. To date, several works exist which use sue according to the protocol described by Roger and Bend- DNA methods for eel identiWcation. Among these meth- ich [23], with slight modiWcations. This is an extraction ods, techniques such as ampliWcation of a partial sequence method based on CTAB and successive washings with phe- of DNA by polymerase chain reaction (PCR) followed by nol and chloroform, which enables to extract puriWed high- direct sequencing of the ampliWed products [8–10], restric- molecular-weight DNA without using expensive equipment tion fragment length polymorphism analysis [11–15], and time-consuming procedures. single nucleotide polymorphism [16, 17], real-time PCR In the case of products used for the methodological vali- analysis [18, 19], microsatellite analysis [20], or randomly dation and analysis of commercial samples, extraction was ampliWed polymorphic DNA analysis method [13, 21, 22] performed using 100–300 mg of tissue. are very useful. All these works include a low number of Quality and concentration were determined by measur- eel species. The work of Rehbein et al. identiWes eel spe- ing the absorbance at 260 nm and the 260/280 nm and 234/ cies products by means of a single-strand conformation 260 ratios using a NanoDrop™ ND-1000 spectrophotometer polymorphism, independent of the treatment applied dur- (Thermo ScientiWc) [24]. DNA extracts were appropriately ing the manufacture, but this study only includes 4 eel labeled and stored at ¡80 °C for subsequent tasks. species [15]. For these reasons, in the present study a genetic method- Primer design, PCR ampliWcation, and DNA sequencing ology based on the ampliWcation of a cyt b fragment is developed. The forensically informative nucleotide Sequences of the cyt b gene were downloaded from the sequencing (FINS) technique is based on a PCR followed database of National Center for Biotechnology Information by a phylogenetic analysis. It represents an accurate and (NCBI) (Table 1) and aligned using BioEdit 7.0 [25]. For reproducible procedure that is not subject to operator bias the aligned sequences, a degenerate primer set was and can be performed independently in any laboratories designed by hand. The forward and reverse primers are, that carry out molecular techniques. This methodology respectively, ANGUILLA_F (5Ј-GGC CGA GGR CTT allows the identiWcation of all eel species included in this TAC TAC GGY-3Ј) and ANGUILLA_R (5Ј- AAT CGG work. GTY ART GTG GCG TTG T-3Ј). To date, there are not any works about the genetic identi- A partial cyt b gene fragment of 239 bp (196 bp with- Wcation of eels which include so many species, regardless out primers) was ampliWed using these primers. All these of the processes used. Here lies the importance of the pres- ampliWcations were carried out in a Wnal volume of 25 L ent work. Due to the great global market of eels, the need containing 100 ng of DNA template, 2.5 L of 10£ PCR V for techniques that take into account a maximum number of bu er, 2 mM MgCl2, 0.8 mM dNTP, 0.8 M solution of species that are susceptible to be marketed as eels arises. each primer, and 0.5 unit of Taq-polymerase (Bioline). The developed tool will be of great help in controlling the All reactions were performed using a Bio-Rad MyCycler correct labeling, improving the protection of consumers’ thermocycler. Conditions for ampliWcation were as fol- rights, and avoiding unfair competition between industry lows: a preheating step of 94 °C for 5 min, followed by 35 operators. cycles of ampliWcation (94 °C for 20 s 52 °C for 20 s and 123 Eur Food Res Technol (2012) 234:689–694 691
Fig. 1 Distribution map of the eel species (Anguilla genus) included A. marmorata, A. megastoma and A. nebulosa; Purple: A. japonica; in the present study. Description of colors: Brown: A. marmorata Black: A. malgumora; Green: A. australis and A. reinhardtii; Pink: and A. megastoma; Blue: A. rostrata; Grey: A. celebesensis and A. dieVenbachii A. megastoma; Yellow: A. anguilla; Orange: A. bicolor, A. celebesensis,
Table 1 Samples belonging to Order Family ScientiWc name Common namea NCBI Anguilla genus included in this work Anguilliformes Anguillidae A. anguilla European eel EU223997 A. australis Short-Wnned eel AB021769 A. bicolor Indonesian shortWn eel AF006708 A. celebesensis Celebes longWn eel AB021777 A. dieVenbachii New Zealand longWn eel AF006711 A. japonica Japanese eel AF006702 A. malgumora Indonesian long-Wnned eel AP007238 A. marmorata Giant mottled eel EF690363 A. megastoma Polynesian long-Wnned eel AB021771 A. nebulosa Mottled eel AP007246 a Only one of the possible A. reinhardtii Speckled longWn eel AF006707 common names for each A. rostrata American eel AP007249 species is shown
72 °C for 20 s) and a Wnal extension step of 7 min at the primer sequences were excluded from the sequencing 72 °C. data. Moreover, DNA sequences from diVerent databases PCR products were sequenced in both directions using were included in the alignment. the same primers of PCR ampliWcation, to avoid sequenc- ing errors. Both strands were sequenced on an ABI Prism Development of the FINS methodology 3130 DNA Genetic Analyzer (Applied Biosystems) using BigDye Terminator Cycle Sequencing Ready Reaction Kit The sequences herein obtained and those downloaded from version 1.1 (Applied Biosystems), following the manufac- GenBank database (Table 1) were used to carry out the turer’s instructions. Nucleotide sequences obtained were phylogenetic analyses. corrected using Chromas 1.45 [26] and subsequently These phylogenetic analyses were carried out using the aligned using ClustalW [27] available in the program Bio- software Mega 4.0, and the genetic distances between the Edit 7.0 [25]. The alignments were corrected by hand, and obtained sequences and those obtained from the GenBank 123 692 Eur Food Res Technol (2012) 234:689–694 database were estimated using the Tamura–Nei model [28]. The inference of the phylogenetic tree has been drawn using the neighbor-joining method [29]. The reliability of the clades formed at the species level in the tree was evalu- ated by means of a bootstrap test with 2.000 replications.
Methodological validation
The aim of the methodological validation was to check whether the manufacturing process which processed food underwent had no inXuence on the detection of eel species. Fig. 2 PCR products obtained from all eel species included in this Individuals from diVerent eel species were authenticated study (Size obtained: 239 bp). Lane 1: 50 bp DNA Ladder; Lane 2–13: on the basis of their morphological traits. Eels are commer- Eel species, in order, Anguilla anguilla, A. australis, A. bicolor, A. cel- ebesensis, A. dieVenbachia, A. japonica, A. malgumora, A. marmora- cialized mainly as fresh or canned. The treatment applied to ta, A. megastoma, A. nebulosa, A. reinhardtii, A rostrata; Lane 14– canned samples involved 121 °C of temperature and 1.2 bars 15: Products from a reagent blank and from a negative control of PCR of overpressure, and diVerent kinds of sauces and condiments reaction where no DNA was added were used. These treatments were carried out in the pilot plant of CECOPESCA (Technical Centre for the preservation of Fish and Aquaculture products) (Spanish National Fish This technique is becoming increasingly widespread, and Seafood Canners Association—Spanish Technical Cen- and its cost is decreasing. However, the main drawback that tre for Fish and Seafood Products Preservation). arises is the acquisition of equipment, which has a high cost Then, the products were analyzed in the same way as the and requires a large investment. On the other hand, highly standard species, using the methodology developed in the qualiWed personnel are necessary to carry it out. present work. Results obtained using morphological fea- In this work, obtained PCR products from ampliWcation tures were compared with those obtained by means of the and sequencing of a cyt b gene fragment of the DNA with application of the methodologies developed to determine the primers herein named generated an amplicon of 196 bp the speciWcity of the methods. Coincidence percentage in all the species included in the present study (Table 1 and between identiWed species on the basis of morphological Fig. 2). The obtained sequences were deposited in Gen- traits and the genetic methodology developed was calcu- Bank under the following accession numbers: JQ312081– lated to establish this speciWcity. JQ312113. This approach is useful to assess the functioning of Genetic distances between these sequences were esti- methodology and to optimize the conditions of the devel- mated using the Tamura–Nei method [27, 33], and a phylo- oped methodology since diVerent treatments applied to genetic analysis was carried out. From the distance matrix, some canned products can degrade the DNA and result in one phylogenetic tree was constructed using the neighbor- PCR inhibitors. The presence of additives such as spices or joining method. sauces used in the alimentary industry attenuates or inclu- The phylogenetic tree constructed from 196-bp sively inhibits the DNA ampliWcation and aVects the qual- sequences shows that all the sequences belonging to indi- ity and quantity of the extracted DNA [30, 31]. viduals of the same species are grouped in the same cluster (Fig. 3), allowing the unequivocal identiWcation of all eel species included in the present work. Three clearly distinct Results and discussion branches in the tree are highlighted. There is no relation between this genetic separation and their geographical dis- The FINS technique described by Bartlett and Davidson [32] tribution. Bootstrap values higher than or equal to 70 usu- was used in the present study to develop an identiWcation ally correspond to a probability higher than or equal to 95% method for eel species. This technique allows the genetic iden- that the corresponding cluster is real [34], giving a quantita- tiWcation of species using phylogenetic analysis of DNA tive measurement of certainty of the assignment of a sam- sequences and is based on the comparison between sequences ple to a particular species. In the present study, the lower of pattern species and sequences of unknown samples. For the bootstrap value was 91, demonstrating the great robustness application of this method, it was necessary to obtain the pat- of the technique that allows the reliable identiWcation of all tern of sequences belonging to the species studied in the pres- species studied. ent work. The inconvenience of the intraspeciWc variability is Methodological validation of results obtained by means of overcame using this method. Also, this method includes a high the application of the method herein developed was in agree- number of species that are not studied thus far. ment with the validation of those based on morphological 123 Eur Food Res Technol (2012) 234:689–694 693
92 Anguilla marmorata for a critical status of eel in Iberian waters. Arch Hydrobiol 144:245–253 5. Moriarty C (1996) The decline in catches of European elver of 91 Anguilla bicolor 1980–1992. Arch Pol Fish 4:245–248 96 Anguilla reinhardtii 6. Boubee JA, Mitchell CP, Chisnall BL, West DW, Bowman EJ, 96 Anguilla japonica 99 Haro A (2001) Factors regulating the downstream migration of 15 Anguilla nebulosa mature eels (Anguilla spp,) at Aniwhenua Dam, Bay of Plenty, 12 91 Anguilla celebesensis New Zealand. N Z J Mar Freshw Res 35:121–134 99 Anguilla australis 7. EU. Council Regulation (EC) No 1100/2007 of 18 September 2007 99 31 Anguilla megastoma establishing measures for the recovery of the stock of European eel 99 Anguilla malgumora 8. Minegishi Y, Aoyama J, Inoue JG, Miya M, Nishida M, Tsukam- 97 Anguilla dieffenbachii oto K (2005) Molecular phylogeny and evolution of the freshwater 98 11 Anguilla anguilla eels genus Anguilla based on the whole mitochondrial genome Anguilla rostrata sequences. Mol Phylogenet Evol 34:134–146 91 9. Jamandre BWD, Shen KN, Yambot AV, Tzeng WN (2007) Molecular phylogeny of Philippine freshwater eels anguilla spp. 0.01 (Actinopterygi: Anguilliformes: Anguillidae) inferred from mito- chondrial. RaZes Bull Zool 14:51–59 Fig. 3 Neighbor-Joining tree showing the relationships 20 among the 10. Sezaki K, Itoi S, Watabe S (2005) A simple method to distinguish studied species, carried from the alignment of 239 bp of the cyt b gene (fragment of 196 bp without primers) two commercially valuable eel species in Japan Anguilla japonica and A-anguilla using polymerase chain reaction strategy with a species-speciWc primer. Fish Sci 71:414–421 W 11. Aoyama J, Watanabe S, Nishida M, Tsukamoto K (2000) Discrim- characters. Therefore, this method shows a speci city of ination of catadromous eels of genus Anguilla using polymerase 100% when it is applied to this kind of products. chain reaction-restriction fragment length polymorphism analysis of the mitochondrial 16S ribosomal RNA domain. Trans Am Fish Soc 129:873–878 12. Hwang DF, Jen HC, Hsieh YW, Shiau CY (2004) Applying DNA Conclusions techniques to the identiWcation of the species of dressed toasted eel products. J Agric Food Chem 52:5972–5977 Altogether, this work describes the development of a 13. Kim WJ, Kong HJ, Kim YO, Nam BH, Kim KK (2009) Develop- method for the authentication of eel species, which could ment of RAPD-SCAR and RAPD-generated PCR-RFLP Markers for IdentiWcation of Four Anguilla eel Species. Animal Cells Syst be used in all molecular laboratories independent of the 13:179–186 equipment available in these laboratories. FINS methodol- 14. Lin YS, Poh YP, Lin SM, Tzeng CS (2002) Molecular techniques ogy is a reliable and reproducible procedure that is based on to identify freshwater eels: RFLP analyses of PCR-ampliWed DNA W phylogenetic analysis of DNA sequences, whose possible fragments and allele-speci c PCR from mitochondrial DNA. Zool Stud 41:421–430 applications are the normative control of raw and processed 15. Rehbein H, Sotelo CG, Perez-Martin RI, Chapela-Garrido MJ, products, particularly the authenticity of imported species; Hold GL, Russell VJ, Pryde SE, Santos AT, Rosa C, Quinteiro J, the veriWcation of the traceability of diVerent Wshing Rey-Mendez M (2002) DiVerentiation of raw or processed eel by batches along the commercial chain; correct labeling; pro- PCR-based techniques: restriction fragment length polymorphism analysis (RFLP) and single strand conformation polymorphism tection of the consumer’s rights; fair competence among analysis (SSCP). Eur Food Res Technol 214:171–177 Wshing operators; and the Wsheries’ control. 16. Frankowski J, Bastrop R (2009) IdentiWcation of Anguilla anguilla (L.) and Anguilla rostrata (Le Sueur) and their hybrids based on a Acknowledgments We thank Mar Huertas (Algarve University of diagnostic single nucleotide polymorphism in nuclear 18S rDNA. Portugal), Dalius Butkauskas (Nature Research Center of Lituania), Le Mol Ecol Resour 10:173–176 Quang Dung (Institute of Marine Environment and Resources of Viet- 17. Itoi S, Nakaya M, Kaneko G, Kondo H, Sezaki K, Watabe S nam), Caroline Coté (Canada), David Cairns (Department of Fisheries (2005) Rapid identiWcation of eels Anguilla japonica and Anguilla and Oceans of Canada), Yves de Lafontaine and Simon Despatie anguilla by polymerase chain reaction with single nucleotide poly- (Aquatic Ecosystems Division. Environnement of Canada). morphism-based speciWc probes. Fish Sci 71:1356–1364 18. Minegishi Y, Yoshinaga T, Aoyama J, Tsukamoto K (2009) Species identiWcation of Anguilla japonica by real-time PCR based on a sequence detection system: a practical application to References eggs and larvae. Ices J Marine Sci 66:1915–1918 19. Watanabe S, Minegishi Y, Yoshinaga T, Aoyama J, Tsukamoto K 1. Hardy JD (1978) Development of Wshes of the Mid-Atlantic Bight: (2004) A quick method for species identiWcation of Japanese eel an atlas of Egg, Larval, and Juvenile stages. Anguillidae thorough (Anguilla japonica) using real-time PCR: An onboard application Syngnathidae. In: Fish and Wildlife Service. Department of the for use during sampling surveys. Mar Biotechnol 6:566–574 Interior, U.S., p 455 20. Maes GE, Pujolar JM, Raeymaekers JAM, Dannewitz J, Volckaert 2. Dekker W (2003) On the distribution of the European eel (Anguil- FAM (2006) Microsatellite conservation and Bayesian individual la anguilla) and its Wsheries. Can J Fish Aquat Sci 60:787–799 assignment in four Anguilla species. Marine Ecol Prog Ser 3. FAO (2003) Fishery and aquaculture statistics 319:251–261 4. Lobon-Cervia J (1999) The decline of eel Anguilla anguilla (L.) in 21. Lehmann D, Hettwer H, Taraschewski H (2000) RAPD-PCR a river catchment of northern Spain 1986–1997. Further evidence investigations of systematic relationships among four species of 123 694 Eur Food Res Technol (2012) 234:689–694
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123
Authentication of gadoids from
highly processed products
susceptible to include species
mixtures by means of DNA
sequencing methods
Página 99 de 153
Eur Food Res Technol (2013) 236:171–180 DOI 10.1007/s00217-012-1875-8
ORIGINAL PAPER
Authentication of gadoids from highly processed products susceptible to include species mixtures by means of DNA sequencing methods
Fa´tima C. Lago • Juan M. Vieites • Montserrat Espin˜eira
Received: 27 July 2012 / Revised: 8 November 2012 / Accepted: 14 November 2012 / Published online: 29 November 2012 Springer-Verlag Berlin Heidelberg 2012
Abstract Economic importance of gadoids such as fish- Keywords Gadoids Genetic identification FINS ing resource, and their conservation status, necessitates the SNP Species mixtures Highly processed products development of techniques that allow unequivocal authentication authentication of products made from them. Amplification of a fragment of mitochondrial cytochrome b (cyt b) marker and subsequent phylogenetic analysis were carried out to Introduction authenticate these products and assure their correct label- ing. Also, SNP analysis that allows detection of species The Gadidae family is one of the most important of all mixtures of Gadus genus was developed. For this, two commercial fish families throughout the world. Their high fragments of the cyt b gene were amplified and sequenced, exploitation levels and their increased worldwide con- one of 464 bp and another internal fragment to this of sumption have provoked a stock decrease and catch 263 bp to allow the authentication of gadoid species in limitations. highly processed products. Obtained sequences were Overfishing data for these species exist from 1950 and aligned and analyzed in order to assess the presence of catastrophic decrease in the volume of their stocks have informative variable positions and a maximum of 14 SNP been detected between 1970 and 2000 [1]. In this sense, were identified and selected. These allow detection and Atlantic cod (Gadus morhua, Linneo 1752) is a species of identification of species mixtures belonging to this genus. this family on which this problem is more acute. For this The developed methodologies were validated and applied reason, international union for the conservation of nature to 25 commercial samples. The main novelty of this work and natural resources (IUCN) has included it in the red list lies in the fact that is the only work that allows the of threatened species (also known as the IUCN red list or detection of species mixtures of the Gadus genus and is the red data list). only one that allows the authentication of highly processed Economic importance of this fishing resource and its products up to date. Furthermore, this methodology allows conservation status forced to the authorities of European identifying more of 15 species of gadoids and can be Union (EU) to establish the total allowable catches (TAC) applied to all kinds of seafood products. Therefore, this in 1970. However, the illegal, unregulated and unreported molecular tool can be applied in questions related to check fishing (IUU) could make that the checking of the fulfill- the fulfillment of labeling regulations for seafood products, ment of these regulations to be difficult. verify the correct traceability in commercial trade and for This situation has brought to the search and capture of fisheries control. alternative species, increasing their number in the market to thus attending to demand of the consumers. Different closer fishes to Atlantic cod are commercialized as if they were F. C. Lago J. M. Vieites M. Espin˜eira (&) this species, especially Pacific cod (Gadus macrocephalus), Research Department of Genomics and Proteomics Applied Greenland cod (Gadus ogac), Ling (Molva molva), Blue to the Marine and Food Industry, ANFACO-CECOPESCA, ling (Molva dypterygia), Pollack (Pollachius pollachius), 36310 Vigo, Pontevedra, Spain e-mail: [email protected] Saithe (Pollachius virens), Haddock (Melanogrammus 123 172 Eur Food Res Technol (2013) 236:171–180 aeglefinus), Alaska pollack (Theragra chalcogramma), mixture detection of different sardine species in fish Blue whiting (Micromesistius poutassou) and Whiting products [27]. (Merlangius merlangus) because they present similar The aim of this work was to develop a methodology for organoleptic and morphological characteristics. These the correct identification of the main species of gadoid, species can be found in the markets in different commercial among which include more of 15 cod species and related formats as, for example, whole, filleted, in cubes, shredded, species, in any format of marketing which can be found in tails,… and including different methods for conservation as the market, and regardless of the degree of transformation fresh, frozen, smoked, in surimi, canned, ready-to-serve to which they have been undergone. In addition, a meth- dishes. odology based on SNP analysis of Gadus species that The increase of processed products and international allows detecting mixing of these species in seafood prod- trade can cause the deliberate or unintentional substitution ucts has been designed too. These tools allow the verifi- of species. The fish species identification is an important cation of the correct labeling of the products. issue to have accounted in regarding to their correct labeling. Labeling regulations for products derived from fisheries are each time more demanding [2, 3]. These leg- Materials and methods islations indicate the necessity of labeling the fish products with both commercial and scientific denomination, in order Sample collection, storage and DNA extraction to assure the traceability along the chain and thus avoid possible fraud. Authenticated specimens of gadoids were collected from Due to above reasons, and to that the species identification different marine locations around the world. When it was is possible in whole individuals based on morphological possible, the individuals were identified on the basis of characteristics but is impossible in processed products, it is morphological traits according to different bibliographic necessary to develop methods that permit the doubtless references [15, 28]. In other cases, authenticated ethanol- identification of the species present in all kinds of processed preserved fish tissues were provided by universities and products. In this context, different techniques that permit the research centers located around the world. Samples were species identification may be used to protect the rights of the labeled after arriving at the laboratory and preserved at consumers, and at the same time also allow a loyal and honest -80 C (Table 1). Moreover, 25 seafood products labeled competition in the fishing industry. Specifically, molecular as cod were provided by import industries or purchased in biology techniques provide a valuable tool to detect labeling supermarkets and shops from Spain in order to apply the mistakes in fishing products. developed methodology. For the cited reasons, DNA-based methods have proven to Genomic DNA was extracted from 30 mg of muscle in be most suitable for species identification in all types of fresh and frozen samples, and from between 100 and products, independently of the manufacturing processes to 300 mg in the case of highly processed products according which have been subjected. Among all of them, the one most to the method described by Herrero et al. [29]. frequently used for the study of gadoids is the PCR–RFLP The quality and concentration of the extracted DNA [4–10], RT-PCR [11, 12] and sequencing analysis [13–15]. were determined by measuring the absorbance at 260 nm The FINS (forensically informative nucleotide and the 260/280 nm and 234/260 ratios using a Nano- sequencing) technique allows the genetic identification of DropTM ND-1000 spectrophotometer (Thermo Scientific). species by means of polymerase chain reaction (PCR) DNA extracts were appropriately labeled and stored at followed by a phylogenetic analysis, using a DNA -20 C for subsequent tasks. sequence database. The main advantage of this technique is that uses the information of all nucleotide positions Amplification and Sequencing of the PCR Products amplified, allowing the genetic identification successfully of a long number of species. A fragment of 464 bp which includes a partial region of the A novel genetic technique applied to the control of fish glutamic acid tRNA and a partial region of cyt b was traceability, and more specifically in gadoids, is the SNP amplified using the primers L14735 (50 AAA AAC CAC (single nucleotide polymorphisms) analysis. This technique CGT TGT TAT TCA ACT A 30) and H15149AD (50 CCI has been used, among other utilities, to make genetic CCT CAR AAT GAY ATT TGT CCT CA 30) described by linkage maps [16–18], in population studies [19–23], and Burgener [30]. PCRs were performed in a final volume of mainly in species identification [24–26]. The technique is 50 lL containing 100–300 ng of DNA template, 5 lL 10X based on the identification of polymorphisms which be buffer, 2 mM MgCl2, 0.8 mM dNTP mix (Bioline), 0.8 lM repeated with a significant frequencies in the gene solution of each primer (Sigma), 1 unit of Taq DNA sequence. This technique has been successfully used in polymerase (Bioline) and molecular biology grade water 123 Eur Food Res Technol (2013) 236:171–180 173
Table 1 Samples included in this work and location of collection Order Family Species Common namea Samplesb Location
Gadiformes Gadidae Gadus morhua Atlantic cod 30 UK, SW, NO, Bay of Biscay, IC, FR, CA, AT Gadus macrocephalus Pacific cod 15 JP, PN Gadus ogac Greenland cod 15 CA, AT Merlangius merlangus Whiting 10 UK, ES, PT, PO, NO, ANE Pollachius pollachius Pollack 10 UK, NO, Cantabrian Sea Pollachius virens Saithe 10 NO, AE Melanogrammus aeglefinus Haddock 7 ES, North Sea Theragra chalcogramma Alaska pollack 11 PN, CN, CA Gadiculus argenteus Silvery cod 2 IC Micromesistius poutassou Blue whiting 12 UK, ES, M, Cantabrian Sea, CA Trisopterus esmarkii Norway pout 3 IC, UK, NO Lotidae Brosme brosme Brismak 6 IC, UK, AT Molva molva Ling 14 IC, ES, PT Molva dypterygia Blue ling 8 IC, ES, PT, ANE Enchelyopus cimbrius Fourbeard rockling 2 IC Lota lota Burbot 9 FI, NO, ANE Phycidae Phycis blennoides Greater forkbeard 2 IC, PT AE Atlantic Eastern, ANE Atlantic Northeast, AR Argentina, AT Atlantic, CA Canada, CN China, ES Spain, FI Finland, FR France, IC Iceland, JP Japan, NO Norway, PN Pacific North, PO Poland, PT Portugal, SW Sweden, UK United Kingdom, US United States a Only it is shown one of the possible common names for each species b Each sample included between 3 and 10 individuals
(Ambion Nuclease-free water) up to adjust to the final using the sequence analysis software v 5.3.1 (Applied volume. The PCR amplifications were carried out in a Bio- Biosystems), and these sequences were analyzed with Rad MyCycler thermocycler, and the cycling conditions Chromas 1.45 software and aligned with Clustal W, were a preheating step of 3 min at 96 C, followed by 24 available in the program BioEdit 7.0 [31, 32]. cycles each of which comprises 20 s at 96 C, 15 s at From them, a new degenerated reverse primer, GADI 50 C and 2 min at 60 C. DOSPEQ_R (50-CCR TAR TTT ACA TCA CGR CAG PCR products were loaded and visualized in 2 % aga- AT-30), was designed by hand for analyses of highly pro- rose gels (Scharlau) in 0.5X TBE buffer (Sigma), using a cessed products. Amplifications of a partial cyt b gene 100 bp DNA ladder (Invitrogen) as the molecular weight fragment of 263 bp (217 bp without primers) were carried standard. Next, double-stranded DNA products were puri- out using this primer with the L14735 primer. PCR fied using Wizard SV Gel and PCR Clean-Up System amplifications, visualization of PCR products and (Promega) according to the instructions of the manufac- sequencing were carried out as described above but using turer. The concentration and purity were measured by the new reverse designed primer. means of a NanoDropTM ND-1000 spectrophotometer The specificity of sequences obtained from both cyt b (Thermo Scientific) as described for DNA extraction. fragments has been evaluated by the basic local alignment Subsequently, sequencing reactions of both DNA search tool (BLAST) in the database of the national center strands were carried out with the primers described previ- for biotechnology information (NCBI)[33]. ously in a final volume of 10 lL and using the BigDye Terminator cycle sequencing kit v 1.1 (Applied Biosys- Development of FINS methodology tems), in an automatic DNA Genetic Analyzer (ABI Prism 3130 Genetic Analyzer) following the recommendations of The FINS technique described by Bartlett and Davidson the manufacturer. Thermal cycle and the concentrations of has been used in the present study to develop an identifi- the sequencing reaction and the subsequent cleanup of the cation method for cod and related species [34]. This sequencing products by ethanol precipitation were carried technique is based in the comparison of sequences of out in accordance with the instructions of the manufacturer unknown samples with sequences belonging to pattern (Applied Biosystems). Finally, raw data were analyzed specimens.
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Two phylogenetic analyses were carried out. The first of process combines two effects: on the one hand, salting and them was carried out from sequences of the partial cyt b drying steps, and on the other hand, the effect of temper- gene (414 bp) and the second, from the sequences obtained ature. The temperature corresponding to smoking of the from the amplification of the internal fragment herein fillets was raised until 121, and 60 C was reached inside of designed (217 bp). These phylogenetic analyses were car- the product. The cooking time depended on the thickness ried out using the software Mega 4.0 using the Tamura–Nei of the fillets. All these treatments were carried out in the model to calculate the genetic distances between sequences CECOPESCA (Spanish National Centre of Fish Processing [35]. The inference of the phylogenetic trees was carried Technology) pilot plant. The products were analyzed with out with the Neighbor-Joining method [36]. The reliability methodologies developed in the present work. of the clades formed at the species level in the trees was The coincidence percentage between the species iden- evaluated by means of a bootstrap test with 2,000 tified on the basis of their morphological traits and genetic replications. methodology developed was calculated to establish the specificity of the method. Design Single Nucleotide Polymorphism (SNP) Analysis Systems Application to commercial products
Cyt b sequences from both fragments obtained from the After the validation of the method developed in the present three species belonging to the Gadus genus (G. morhua, work, this was applied to 25 products labeled as cod in G. macrocephalus and G. ogac) were aligned with Clustal order to identify the species from which seafood products W, included in BioEdit 7.0 and edited to generate reliable were made and determine the existence of species mixture consensus sequences for each species [31]. For each posi- of the Gadus genus in processed products (Table 3). These tion in both alignments obtained, if a consensus could not products were acquired in Spanish supermarkets, and the be found, an X was used to indicate ambiguity. By com- purpose of these analyses has been to evaluate the situation paring the consensus of each species in both alignments, regarding the labeling of these products on the market. nucleotide differences (SNP) were identified. In the same way, the determination of the limit of detection (LOD) of the methodology developed was Results and discussion established from DNA dilutions and tissue mixtures of specimens belonging to the Gadus genus (G. morhua, Amplification and sequencing of PCR products G. macrocephalus and G. ogac). In the first case, the range of extracted DNA from species of Gadus genus varied In this work, the cyt b gene was evaluated to design a between 250 ng/lL and 1 ng/lL. The dilutions were pre- method that allows the identification of more of 15 cod pared by adding DNA from G. macrocephalus/G. ogac to species and related species, and the detection of species the G. morhua DNA until completing the final amount. mixture of the Gadus genus in highly processed products In the second case, the mixtures of G. macrocephalus/ by means of amplification, phylogenetic analysis and G. ogac/G. morhua were prepared using percentages from subsequently SNP analysis. 100 to 0 % of tissue. The DNA extractions were performed A 464 bp fragment was generated from DNA amplifi- from these tissue mixtures to evaluate the minimum ratio cation with the L14735/H15149AD primers in all of sam- G. macrocephalus/G. ogac/G. morhua that can be detected ples included in the present study (except a 463 bp with the developed method. fragment to Molva molva and 465 bp to Phycis blenno- ides). The sequences were submitted to NCBI database Methodological validation (accession numbers: KC128863-KC128879). There are coincidence between the species identified on At least one of all individuals of the different species the basis of morphological traits and the result evaluated by included in this study was authenticated on the basis of the BLAST in the database of the NCBI. their morphological traits (Table 1). Then, the main treat- In order to allow the authentication of gadoid species in ments applied to commercial products were applied to highly processed products, other methodological strategy them. These were salting, smoking, marinating and pre- was developed. In fresh or frozen fish, it is possible to cooking. For each one, different kinds of sauces and con- amplify the fragment of 464 bp but, in the case of products diments were used (Table 2). The same treatments were undergoing to drastic treatments, it is not possible because applied to mixtures of G. morhua with G. macrocephalus the thermal treatment and pressures generate DNA frag- and G. ogac to check the sensitivity of the developed mentation and this difficult or even impossibility the method. Among the treatments applied, the smoking amplification of this fragment [27, 37–41]. For this reason, 123 Eur Food Res Technol (2013) 236:171–180 175
Table 2 Samples processed Types of processing Product Species included and analyzed for validation Frozen Fillets G. morhua G. macrocephalus G. ogac G. morhua/G. macrocephalus/G. ogac Salted Fillets G. morhua G. macrocephalus G. ogac G. morhua/G. macrocephalus/G. ogac Tails G. morhua G. macrocephalus G. ogac G. morhua/G. macrocephalus/G. ogac Loins G. morhua G. macrocephalus G. ogac G. morhua/G. macrocephalus/G. ogac Small pieces of cod G. morhua G. macrocephalus G. ogac G. morhua/G. macrocephalus/G. ogac Smoked Fillets G. morhua G. macrocephalus G. ogac G. morhua/G. macrocephalus/G. ogac Precooked Cod G. morhua G. macrocephalus G. ogac G. morhua/G. macrocephalus/G. ogac Peppers stuffed with cod G. morhua G. macrocephalus G. ogac G. morhua/G. macrocephalus/G. ogac Salted cod G. morhua G. macrocephalus G. ogac G. morhua/G. macrocephalus/G. ogac Marinate G. morhua G. macrocephalus G. ogac G. morhua/G. macrocephalus/G. ogac Cod sticks G. morhua G. macrocephalus G. ogac G. morhua/G. macrocephalus/G. ogac Cod croquettes G. morhua G. macrocephalus G. ogac G. morhua/G. macrocephalus/G. ogac
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Table 3 Commercial samples analyzed Products Species labeled Species identified by Presence G. FINS/SNP Analysis morhua RT- PCRa
Salted cod tails Gadus morhua Gadus macrocephalus/ogac Negative Gadus morhua Gadus morhua Positive Gadus macrocephalus Gadus macrocephalus/ogac Negative Frozen cod fillet Gadus morhua Gadus morhua Positive Gadus morhua Gadus morhua Positive Frozen cod fish Gadus morhua Gadus morhua Positive Gadus morhua Gadus morhua Positive Salted cod loins Gadus morhua Gadus morhua Positive Gadus macrocephalus Gadus macrocephalus/ogac Negative Battered cod loins Gadus morhua Gadus morhua Positive Gadus morhua Gadus morhua Positive Cod fillets Gadus morhua Gadus morhua Positive Cod sticks Gadus spp Gadus macrocephalus/ogac Negative Gadus morhua Gadus morhua Positive Gadus spp Gadus morhua Positive Gadus morhua Gadus morhua Positive Smoked cod roe Gadus morhua Gadus macrocephalus/ogac Negative Gadus macrocephalus Gadus macrocephalus/ogac Negative Gadus macrocephalus Gadus macrocephalus/ogac Negative Cod head Gadus spp Gadus morhua Positive Cod cheeks Gadus spp Gadus morhua Positive Gadus spp Gadus morhua Positive Gadus spp Gadus macrocephalus/ogac Negative Salted little pieces of cod Gadus spp G.morhua and G. macrocephalus/ogac Mixture Gadus spp G.morhua and G. macrocephalus/ogac Mixture a Positive result: amplification pattern showed by Atlantic cod species, with Ct values about 19 ± 0.5; negative result: amplification pattern showed by other species with Ct values [30; mixture: Ct values 25.9 and 27.2 in order to carry out the genetic identification in these 100 Trisopterus esmarkii products, a 263 bp fragment was amplified (except 262 bp 100 fragment to Molva molva and 265 bp to Phycis Gadus ogac/macrocephalus 99 Gadus morhua 97 blennoides). Theragra chalcogramma 99 99 Merlangius merlangus Melanogrammus aeglefinus Development of FINS methodology 100 100 Pollachius pollachius 99 Pollachius virens 100 Phylogenetic analysis based on the sequences of both 100 Gadiculus argenteus fragments of the cyt b gene was carried out separately, Micromesistius poutassou 100 allowing the establishment of the relationships between Enchelyopus cimbrius 100 100 gadoid species by means of the construction of phylogenies Molva dypterygia 93 Molva molva 100 using these two data sets (Figs. 1, 2). 100 For each fragment, as 464 bp as 263 bp fragment, the Lota lota Brosme brosme 100 genetic distances between the cyt b gene sequences of all Phycis blennoides the studied species were estimated using the Tamura–Nei 100 method [42]. From the two distance matrices of the cyt b, 0.02 two phylogenetic trees were constructed using the Neigh- bor-Joining method (Figs. 1, 2). Fig. 1 Neighbor-Joining tree showing the relationships between the studied species, carried from the alignment of 464 bp of the cyt b Both phylogenetic trees show that the species belonging gene (fragment of 413 bp without primers). The bootstrap values are to the same family are grouped. These are the families located above nodes
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99 Trisopterus esmarkii these species have minor commercial interest than Atlantic 75 Gadus ogac/macrocephalus cod (G. morhua)[46]. 96 Gadus morhua 83 In conclusion, the proposed strategy allows the ampli- 99 Theragra chalcogramma 81 Merlangius merlangus fication of a DNA fragment sufficiently long to discrimi- Melanogrammus aeglefinus 99 98 Pollachius pollachius nate successfully all the gadoid species included in this 80 Pollachius virens work, even in seafood products in which the DNA could be 99 Micromesistius poutassou 99 highly degraded and which may contain species mixture. Gadiculus argenteus 99 Enchelyopus cimbrius 99 99 Molva dypterygia Design single nucleotide polymorphism (SNP) analysis 93 Molva molva 97 99 Lota lota for the detection of mixed species of the genus Gadus Brosme brosme 99 Phycis blennoides 99 From both fragments, consensus sequences from the dif-
0.02 ferent species belonging to the Gadus genus (G. morhua, G. macrocephalus and G. ogac) were built. Fig. 2 Neighbor-Joining tree showing the relationships between the From 464 bp fragment of the cyt b gene, 14 different studied species, carried out from the alignment of 263 bp of the cyt b gene (fragment of 217 bp without primers). The bootstrap values are SNP highly conserved (presents in all analyzed samples) located above nodes were identified, and 7 of which belong to the internal fragment amplified (263 bp). All mutations were transi- Gadidae, Lotidae and Phycidae, all of them included in this tions, with one exception, and involved C/T (92.3 %) and work. Also, worth noting that all the species belonging to the G/A changes (Table 4). According to Kocher et al. [47] same genus have been grouped into the same branch, and that who showed that within a particular species, and also all the sequences belonging to individuals of the same spe- between closely related species, transitions are more cies have been grouped in the same cluster and are well common than transversions, transversional mutations were differentiated, allowing their identification to species level. most frequent in the more distantly related taxa. All Into the phylogenetic tree created from the 413 bp mutational events have occurred at third positions and were fragment, all branches at species level have a bootstrap synonymous. value higher than 97, and in the phylogenetic tree created The existence of heterozygous for each SNP diagnostic from the 217 bp fragment this value is 75. These values site was informative and has showed mixed of G. morhua/ reflect the reliability of the assignation because it has been G. macrocephalus/G. ogac species. calculated that bootstrap values higher or equal to 70 The BLAST was designed to evaluate the specificity of usually correspond to a probability higher or equal to 95 % sequences. The obtained sequences were included in this that the corresponding cluster is real [43], giving a quan- program and given a measure of sequence similarity of titative measurement of the certainty of the assignment of a 100 %, based on the optimal local alignment between sample to a particular species. sequences. Also, to verify the correct operation of the technique Serial dilutions of genomic DNA extracted from species developed, a BLAST analysis has been made to the of the Gadus genus were tested to assess the sensitivity of sequences obtained. As a result, it is to note that all the the developed methodology. The DNA used was obtained sequences obtained have a high level of homology with both from frozen pattern tissue as of tissue of these which other sequences belonging to the same species and which have been undergoing to typical treatments used in can- are deposited in GenBank. ning. This latter treatment was selected as being the most On the other hand, it is important to note too that Gadus aggressive process to which a product can be subjected for ogac and Gadus macrocephalus are grouped into the same that the DNA be highly degraded. The total DNA quantities clade. This indicates that these fragments from cyt b gene used for the PCRs were obtained by mixing DNA from do not allow differentiating between these two species of G. morhua with decreasing amounts of DNA from the Gadus genus. This result is in accordance with previous G. macrocephalus/G. ogac. The LOD for detection of work such as Calo-Mata et al. [44], who using the same species mixtures of the Gadus genus, employing dilutions fragment, and Carr et al. [45], who studding the molecular of genomic DNA, was 20 ng for all species from frozen divergences between these species and conclude that tissues, and 100 ng, for canning products. Greenland cod (G. ogac) and Pacific cod (G. macroceph- Also, DNA extractions were performed from mixture alus) have essentially identical mtDNA sequences. Other tissues from species of the Gadus genus to estimate the authors do reference to possible genomic differences of optimal amount of tissue necessary to obtain a high sen- subspecies but, finally, this fact is so not important because sitivity. Specifically, the DNA was extracted from 300 mg
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Table 4 Gadus SNP in species G.macrocephalus Mixed species G. morhua / G. ogac Gadus genus GADSNP- 81 C T Y GADSNP- 156 CT Y GADSNP-165 T CY217 bp GADSNP- 222 CT YFragment GADSNP- 228 C T Y GADSNP- 234 AC/TM or W 413 bp GADSNP- 255 TC Y Fragment GADSNP- 273 C T Y GADSNP- 282 TC Y GADSNP- 288 GA R GADSNP- 291 C T Y GADSNP- 315 T CY Y = C ? T; M = A ? C; GADSNP- 318 C T Y W = A ? T; R = G ? A GADSNP- 333 GA R
of mixtures tissues from G. morhua and G. macrocephalus/ All identified species in these samples by means of the G. ogac in different proportions. method herein developed have been in agreement with The diagnostic method herein designed was applied to those based on morphological characters. On the other these mixtures, allowing the establishment of the minimum hand, the species mixtures of the Gadus genus were amount of tissue from G. macrocephalus/G. ogac which detected in all these samples that contained these species can be detected in the conditions previously described. The independently of the transformation process. Therefore, the detection limit was lower than 5 % of tissue from G. methodology shows a specificity of 100 %. macrocephalus/G. ogac using 300 mg for DNA extraction. Application to commercial products The results obtained in the evaluation of specificity and sensitivity of methodology developed show that this tech- The developed methodology was applied to 25 commercial nique is highly reliable. samples labeled as cod. This application allows to knowing The design of a specific method for detection of the the degree of fulfillment of the labeling regulations in these mixture of the 3 Gadus species (G. morhua, G. macro- products and determinate the existence of species mixtures cephalus and G. ogac) was chosen due to they are the most of the Gadus genus in processed products. All commercial similar species both morphologically and genetically. If the samples analyzed were correctly identified in accordance mixture with other species included or even with some with the declared species except 13 of them (8 %), where species not listed in this work had taken place, the genetic the name of the species displayed in the label was not in difference would be so obvious that the species mixture agreement with the species contained (Table 3). would be detected by the appearance of numerous double Therefore, species mixtures of the Gadus genus were peaks in the chromatograms of sequencing. detected in 8 % of the commercial products tested, but Methodological validation these were correctly labeled because they were labeled as ‘‘Gadus spp.’’ and the contained species were not specified. The aim of the methodological validation was to check In the present work, a DNA-based methodology that whether the manufacturing process to which processed allows the genetic identification of the most important food have been undergone, have no influence on the commercialized species of cod and related species, and the identification of all species included in the present work detection of species mixtures of Gadus genus in fish and in the detection of species mixtures of the Gadus products has been developed. It is to note that it is the only genus. one that allows the authentication of over 15 gadoid spe- Different seafood products were prepared in the pilot cies, even in highly processed products, regardless of the plant of CECOPESCA simulating the conditions used in the degree of transformation to which they have undergone. food industry. Among these products are products made The main novelty of this work lies in the fact that is the from each of the species included in this work and products only work that allows the detection of species mixtures of made from species mixture of the Gadus genus. The elabo- the Gadus genus and is the only one that allows the rated products were analyzed by the proposed methodology. authentication of highly processed products up to date. All different transformation treatments applied allowed The developed tool is based on the amplification of a evaluating the correct PCR amplification in these processed fragment of mitochondrial cyt b marker and subsequent products. phylogenetic analysis (FINS) following by SNP analysis.
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The sequencing is a technological process widely used in spp.) and Atlantic cod (Gadus morhua) using PCR-RFLPs, FINS control laboratories, so the technology developed in this and BLAST. J Agric Food Chem 56(22):10865–10871 10. Wolf C, Burgener M, Hubner P, Luthy J (2000) PCR-RFLP study could be easily implemented for the authentication and analysis of mitochondrial DNA: differentiation of fish species. identification technologies, without it mean an increase in Lebenson Wiss Technol 33:144–150 cost. 11. Herrero B, Madrin˜a´n M, Vieites JM, Espin˜eira M (2010) These characteristics are turning it into a very appropriate Authentication of Atlantic cod (Gadus morhua) using Real -Time PCR. J Agric Food Chem 58(8):4794–4799 tool for the authentication of gadoids and detection of species 12. Taylor MI, Fox C, Rico I, Rico C (2002) Species specific Taq- mixtures of the Gadus genus in all kind of seafood products. Man probes for simultaneous identification of (Gadus morhua L.) The possible applications of this method are the following: haddock (Melanogrammus aeglefinus L.) and whiting (Merlan- normative control of raw and processed products, particu- gius merlangus L.). Mol Ecol Notes 2:599–601 13. Bakke I, Johansen SD (2005) Molecular phylogenetics of Gadi- larly for the authenticity of imported species; the verification dae and related Gadiformes based on mitochondrial DNA of the traceability of different fishing batches along the sequences. Mar Biotechnol 7:61–69 commercial chain; correct labeling and protection of the 14. Mora´n P, Garcia-Vazquez E (2006) Identification of highly rights of the consumers; fair competence among fishing prized commercial fish using a PCR-based methodology. Bio- chem Mol Biol Educ 34(2):121–124 operators; and the control of the fisheries. 15. Teletchea F, Laudet V, Hanni C (2006) Phylogeny of the Gadidae (sensu Svetovidav, 1948) based on their morphology and two Acknowledgments We thank Maritza Barriga (Instituto Tecnolo´g- mitochondrial genes. Mol Phylogenet Evol 38:189–199 ico Pesquero del Peru´ (ITP)), Jonbjorn Palson (Marine research 16. Hubert S, Higgins B, Borza T, Bowman S (2010) Development of Institute, Iceland), Kathryn E. Elmer and Axel Meyer (University of a SNP resource and a genetic linkage map for Atlantic cod Konstanz, Germany), Steve Hay (Marine Science Scotland), James (Gadus morhua). BMC Genomics 11:191 Markham and Ann Gorman (Lake Erie Fisheries Unit Dunkirk, New 17. Moen T, Delghandi M, Wesmajervi MS, Westgaard JI, Fjalestad York), and Dawn Roje (School of Aquatic and Fishery Sciences, KT (2009) A SNP/microsatellite genetic linkage map of the University of Washington), Peter Smith (National Institute of Water Atlantic cod (Gadus morhua). 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DISCUSIÓN
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DISCUSIÓN
Entre las técnicas moleculares empleadas para la identificación de especies, la técnica FINS (secuenciación del ADN seguida de análisis filogenético) ha sido de las más utilizadas. Esta metodología se ha aplicado con éxito en la identificación genética de diferentes grupos taxonómicos, entre los que destacan distintos trabajos de escómbridos [6, 50-53], lofioides [24, 54], xífidos [19], salmónidos [13], clupeidos [12, 20, 55-59], gadiformes [60, 61], pleuronéctidos [23], escualos [62], moluscos [22, 30], carnes [63], anguílidos [64], algas [27, 31], pepinos de mar [65], dípteros [66, 67],…
El correcto funcionamiento de las metodologías desarrolladas depende de varios factores, desde la selección de un método de extracción de ADN adecuado, el correcto diseño de cebadores, la optimización de la PCR, el número de especies incluidas, su validación y posterior aplicación a muestras comerciales,...
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EXTRACCIÓN DEL ADN
El aislamiento y purificación del ADN es una etapa de suma importancia en las metodologías de identificación de especies.
Entre la gran variedad de métodos de extracción de ácidos nucleicos, en los trabajos incluidos en esta tesis doctoral se optó por el método de extracción mediante Fenol-Cloroformo desarrollado por Murray y Thompson en 1980, y publicado por Wagner en 1987 [68]. En el caso de las muestras procesadas pertenecientes a los estudios de identificación genética de jureles, sardinas y especies afines (muestras de la validación metodológica y el análisis de muestras comerciales), tras la extracción con Fenol-Cloroformo se realizó, en algunos casos, la purificación de las extracciones utilizando columnas de sílica, ya que los extractos sin purificar contienen en muchas ocasiones inhibidores y no funcionan adecuadamente en la PCR.
Previamente a la extracción con solventes se ha realizado la digestión del tejido con un tampón que incluye CTAB (bromuro de hexadeciltrimetilamonio), que es un detergente con capacidad de digerir polisacáridos y polifenoles que se resisten a otros detergentes, por lo que es adecuado para procesar tejidos y todo tipo de muestras, incluidas productos comerciales.
Una de las ventajas de utilizar estos métodos tradicionales es su bajo coste, así como un alto rendimiento y su fácil estandarización, permitiendo obtener ADN de alta calidad, sin inhibidores que afectan la PCR [69].
La purificación del ADN extraído a partir de productos procesados mediante columnas de sílice, se basa en la capacidad de adsorción de los ácidos nucleicos en estas columnas en presencia de altas
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concentraciones de sales caotrópicas. Los contaminantes de la muestra son eliminados mediante sucesivos lavados y, finalmente, el ADN es eluído en agua o tampón de baja fuerza iónica a pH neutro o ligeramente alcalino. La ventaja de estos métodos es la simplicidad y rapidez de sus protocolos. Es importante no saturar las columnas con ADN ya que el rendimiento y la calidad del ADN obtenido depende en gran medida de ello.
En todos los casos, la concentración del ADN extraído se determinó mediante la absorbancia a 260 nm la pureza mediante las ratios de absorbancia a 260/280 nm y 234/260 respectivamente [70] con un espectrofotómetro NanoDropTM ND-1000 (Thermo Scientific). La cantidad y calidad de ADN obtenida para todas las muestras fue adecuada para las posteriores etapas de análisis.
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SELECCIÓN DEL MARCADOR MOLECULAR
Los avances en la tecnología del ADN han permitido la caracterización de marcadores moleculares muy estables para la identificación de especies. A pesar del hecho de que el análisis de ADN es más complejo que el análisis de proteínas, la gran estabilidad de esta molécula y la gran cantidad de información que ofrece, lo convierte en un marcador molecular poderoso para la identificación de especies.
Los marcadores moleculares basados en el ADN constituyen herramientas poderosas para detectar dichas variaciones en el genoma (mutaciones, inserciones o deleciones). Son empleados en estudios de genética de organismos vegetales, animales, así como de bacterias, parásitos y virus, permitiendo evidenciar variaciones o polimorfismos en la secuencia de ADN de los individuos, modifiquen estas o no su fenotipo. Sus aplicaciones son muy diversas: diferenciación de individuos, discriminación entre clones, análisis filogenéticos y taxonómicos, mapeo de genomas, cuantificación de variabilidad génica intra e interespecífica, mejoras genéticas, detección de infecciones o propensión a sufrirlas, localización de resistencia a enfermedades y dispersión de especies.
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La elección del marcador es fundamental y debe poseer unas características concretas:
Altamente polimórfico, es decir, variable en un grupo de individuos. De herencia mendeliana no epistática (sin interacción entre genes). Codominante, capaz de distinguir entre un homocigoto y un heterocigoto. Reproducible en cualquier experimento de laboratorio. Distribuido de manera uniforme en todo el genoma. Discriminante, capaz de detectar diferencias entre individuos estrechamente relacionados. No sujeto a influencias ambientales. De posible detección en estadíos tempranos del desarrollo.
Los marcadores mitocondriales, debido a sus características, presentan una serie de ventajas respecto a los nucleares para su aplicación en trabajos de identificación genética. El ADN mitocondrial posee una tasa de mutación mayor que el ADN nuclear, por lo que es más indicado para diferenciar especies cercanas. Otra ventaja es el alto número de copias que posee cada célula [6, 71]. Por término medio se estima que hay de 10 a 100 mitocondrias por célula y de 10 a 100 copias de ADN mitocondrial por mitocondria, lo que supone de 100 a 10.000 copias de ADN mitocondrial por célula. Este elevado número de moléculas hace que su recuperación en aquellos casos en los que el ADN de partida es muy escaso o está muy degradado, sea mucho más eficiente que en el caso del ADN nuclear. Se hereda por vía materna en la mayoría de las especies, por lo que presenta ausencia de recombinación (a diferencia de la herencia biparental que presenta recombinación por tener información de ambos progenitores). Pasa intacto entre generaciones
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salvo por las mutaciones; facilitando la identificación de las relaciones entre organismos muy parecidos. Esto permite estudiar las poblaciones hacia atrás en el tiempo, buscando la ascendencia materna de una población [72].
Las regiones genómicas cytb y COI han sido ampliamente utilizadas como marcadores moleculares en estudios de identificación genética de especies [20, 21, 73-75]. Por este motivo, ambos marcadores fueron utilizados en las metodologías desarrolladas en la presente tesis doctoral: un fragmento del gen cytb fue empleado para identificar diferentes especies de carnes, jureles, sardinas, anguilas y gádidos; y un fragmento del gen COI, para el desarrollo de la herramienta molecular de identificación de especies de rayas. En todos ellos el marcador fue seleccionado porque permitió la diferenciación de todas las especies objeto de estudio, así como otras muy relacionas que pueden emplearse como especies sustitutas.
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DISEÑO DE CEBADORES
En la bibliografía actual existen numerosos paquetes informáticos para el diseño de cebadores y sondas, (AlleleID, Beacon Designer o Primer Express). En los estudios de identificación genética de carnes, jureles y especies afines, rayas y anguilas incluidos en la presente tesis doctoral, los cebadores se diseñaron de forma manual, a partir de los alineamientos de secuencias obtenidos del NCBI.
En el caso del trabajo de identificación de especies de sardinas, los cebadores empleados fueron los de Jerome et al. [55, 56].
Para la metodología de identificación de especies de gádidos, se partió de los cebadores diseñados por Burgener et al., L14735 y H15149AD [76]. Tras alinear las secuencias de todas las especies incluidas en este estudio, se diseñó de forma manual un cebador reverso interno a este fragmento de 464 pb, que permite la amplificación de un fragmento de 263 pb.
Una vez diseñados los cebadores, se solicitó su síntesis en distintas casas comerciales con purificación mediante HPLC, ya que evita que las secuencias incompletas formadas durante la síntesis puedan competir con el cebador completo.
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ANÁLISIS FILOGENÉTICO
En los trabajos incluidos en esta tesis se han realizado distintos análisis filogenéticos utilizando métodos de distancias.
El algoritmo utilizado para la construcción del árbol filogenético es el de Neighbor-Joining [9] utilizando el modelo de sustitución nucleotídica de Tamura-Nei [77].
Para cada estudio se ha generado una matriz de distancias genéticas que permiten determinar las similitudes entre las secuencias de las muestras desconocidas y las secuencias de referencia, generando un árbol filogenético.
Además, todos los árboles obtenidos mediante las metodologías desarrolladas para cada uno de los grupos taxonómicos estudiados agrupan las secuencias pertenecientes a individuos de la misma especie en el mismo clado, permitiendo la diferenciación de todas las especies incluidas en el estudio y haciendo posible su identificación.
Mediante el análisis de bootstrap con 2000 repeticiones se ha comprobado la robustez o soporte de los distintos nodos obtenidos a nivel de especie, obteniendo valores mayores o iguales a 70 en todos los casos, que corresponden con una probabilidad igual o mayor al 95% que el nodo sea real [10].
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VALIDACIÓN METODOLÓGICA
El objetivo de la etapa de validación fue verificar que los diversos tratamientos a los que son sometidos los productos en el proceso de transformación, no afectan a la correcta aplicación de la técnica desarrollada.
En todos los trabajos que incluye la presente tesis doctoral se ha llevado a cabo una validación metodológica, donde se evalúa la técnica desarrollada para definir su especificidad, robustez, sensibilidad y repetibilidad. Este paso es esencial para asegurar que la metodología produce resultados válidos y adecuados para los fines previstos. Consiste en aplicar la metodología desarrollada a diferentes productos con los formatos de comercialización que se pueden encontrar en el mercado, pero manufacturados a partir de especies conocidas, previamente autentificadas por distintas técnicas (genética y/o morfológicamente).
La obtención de muestras patrón es uno de los pasos más importantes, más complejos y laboriosos, tanto para el desarrollo de la técnica como para la validación metodológica. En muchos casos no es posible obtener en el mercado una determinada especie, por lo que la colaboración con centros de investigación ha sido fundamental para poder llevar a cabo estos trabajos. Las muestras de tejido consistieron en individuos frescos, congelados o conservados en etanol (éstas últimas procedentes de museos, principalmente). Las muestras fueron autentificadas inicialmente en base a sus características morfológicas, zonas de captura,… utilizando la bibliografía disponible en cada caso. Algunas de ellas fueron utilizadas como materia prima para la elaboración de los distintos productos que representan los principales
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formatos de comercialización existentes y que son utilizados en la etapa de validación.
La preparación de los productos se realizó en la planta piloto de ANFACO-CECOPESCA. Entre los tratamientos más agresivos aplicados destacan las conservas y el ahumado. Para la realización de las conservas se aplicó una temperatura de 121 C y 1,2 bares de presión durante un tiempo variable en función del tamaño de los envases. En cuanto al ahumado se les aplicó una temperatura de 121 C hasta que el interior de la pieza alcanzó una temperatura de 60 C. En este caso, al igual que en el anterior, el tiempo de cocción es dependiente del tamaño del producto. Además, en la elaboración de los productos se añadieron diferentes salsas y condimentos, para evaluar cómo afectan al funcionamiento de los métodos desarrollados [13].
Se calculó el porcentaje de coincidencia entre las especies identificadas inicialmente mediante características morfológicas o secuenciación y la técnica desarrollada en el presente trabajo, obteniéndose en todos los casos coincidencia y, por tanto, un 100% de especificidad.
De este modo las técnicas desarrolladas en cada estudio son aplicables a cualquier tipo de producto, independientemente del grado de procesamiento al que haya sido sometido.
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APLICACIÓN A MUESTRAS COMERCIALES/ESTUDIO DE MERCADO
Hace poco menos de una década, las aplicaciones moleculares tan solo eran utilizadas en los más modernos laboratorios de Centros de Investigación y Universidades, que contaban con una gran dotación de equipamiento científico. Sin embargo, esta situación ha cambiado considerablemente debido a varios factores, entre los que destacan: la bajada del coste del equipamiento y del material fungible utilizado, la extensión de esta tecnología a multitud de campos del conocimiento; y la simplificación de los tediosos protocolos de trabajo existentes inicialmente. Estos avances hicieron posible la llegada de esta tecnología a los laboratorios de control de calidad de los productos alimentarios, y con ello su aplicación a cuestiones diversas.
De forma paralela a estos cambios en la tecnología, también ha habido una transición de las causas que impulsan el desarrollo de las técnicas moleculares, como consecuencia de las exigencias que imponen los consumidores del siglo XXI. Frente a la utilización inicial de estas técnicas como una herramienta para dirimir conflictos entre operadores económicos relacionados con la naturaleza u origen de una determinada materia prima, actualmente estas técnicas son utilizadas como una herramienta que permite garantizar a los consumidores la autenticidad de un determinado producto alimenticio, el cual posee ciertas características que lo diferencian de otros muy semejantes, y por las que el consumidor está dispuesto a pagar un sobreprecio del producto. Se ha pasado de un enfoque inicial basado en la legalidad entre proveedor y comprador, a un enfoque actual marcado por la exigencia del consumidor de productos diferenciados y genuinos, en los que se valoran las cualidades de determinadas materias primas frente a otras muy similares.
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La aplicación de las técnicas moleculares desarrolladas a muestras comerciales permite evaluar la situación del mercado en cuanto a etiquetado. La utilidad principal de estas herramientas es garantizar la trazabilidad y autenticidad de productos alimentarios. Con dichos estudios, se evalúa el correcto etiquetado de los productos a la vez que se verifica que las técnicas propuestas funcionan adecuadamente. Las muestras analizadas deben ser representativas de los principales formatos de comercialización de cada grupo de especies. Por ello, todos los trabajos incluidos en la presente tesis doctoral, exceptuando el de anguilas, contienen una parte de aplicación a muestras comerciales en la que se han analizado los principales formatos de comercialización de cada grupo taxonómico (20 productos elaborados a partir de carnes, 15 de jureles, 83 de sardinas, 20 de rayas y 25 etiquetados como bacalao). Los resultados de estos estudios por grupo taxonómico se resumen a continuación.
Identificación genética de especies cárnicas: Se analizaron 20 productos cárnicos de los que en el 15% de los casos el nombre de la especie indicada en la etiqueta no se correspondió con la identificada mediante el análisis genético. En concreto, 3 muestras estaban incorrectamente etiquetadas como filetes de eland, solomillo de potro y hamburguesa de ternera. El análisis genético demostró que eran filetes de kudu, solomillos de ternera y hamburguesas de cerdo respectivamente. Las 17 muestras restantes (85%) estaban correctamente etiquetadas. Aunque en el estudio desarrollado no se han detectado productos con mezcla de especies, esta situación se haría evidente ante la presencia de dobles picos en los cromatogramas obtenidos tras la secuenciación.
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Identificación genética de jureles y especies afines: La metodología desarrollada se aplicó a 15 productos en conserva etiquetados como jurel. Todos los productos analizados estaban etiquetados con el nombre científico y con la denominación comercial de ”jurel o chicharro”. Este es un problema importante en todos los productos elaborados a partir de jurel, donde una única denominación comercial se aplica a esta familia que incluye un gran número de especies morfológicamente muy similares, pero con distinto valor comercial. Los resultados obtenidos mostraron que todos los productos estaban correctamente etiquetados y, además, que las especies más comunes utilizadas en elaboraciones en conserva son Trachurus murphyi y Trachurus trachurus.
Identificación genética de sardinas y especies afines: El Codex Stan 94 establece que las sardinas en conserva o los productos tipo sardina se preparan con pescado fresco o congelado de una lista de 21 especies, haciendo hincapié en Sardina pilchardus. Esta Norma se aplica a las sardinas en conserva y a los productos tipo sardina en agua, aceite u otros líquidos de cobertura, y no se aplica a otros productos especiales cuando el contenido en pescados sea inferior al 50% del contenido neto del envase. Además, sólo los productos elaborados con pescado de la especie S. pilchardus, pueden comercializarse con el nombre comercial de “Sardinas en conserva”, mientras que los productos elaborados a partir de otras especies se denominan “Sardina X”, debiendo referirse al país, área geográfica, especie o nombre común de la especie, de acuerdo con la ley y costumbre del país donde se vende el producto, y siempre de una manera que no engañe a los consumidores. El principal problema de esta Norma es que es confusa para los consumidores, porque bajo la denominación “Sardina
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X” pueden incluirse varias especies de géneros diferentes, incluso de familias diferentes, pero no todas tienen el mismo valor comercial y la misma calidad. En este sentido, uno de los principales objetivos de esta metodología es que mediante esta técnica se puedan eliminar ciertas dudas que puedan surgir con la ambigüedad del nombre “Sardinas X”. La metodología desarrollada se aplicó a 83 muestras comerciales, todas conservas, de las que un 84,38% (70 muestras) fueron correctamente identificadas como S. pilchardus, pero 13 de ellas (15,66%) resultaron estar incorrectamente etiquetadas. De estas 13 muestras, 10 etiquetadas como sardina en conserva contenían S. aurita en lugar de S. pilchardus, y otras 2, S. longiceps en lugar de S. pilchardus. Además, una muestra contenía mezcla de S. aurita y S. pilchardus. Los resultados mostraron que las especies más comunes utilizadas como especies sustitutas en las sardinas en conserva son S. aurita y S. longiceps.
Identificación genética de rayas: El método diseñado fue aplicado a 20 muestras etiquetadas como rayas. Todos los productos elaborados a partir de raya analizados estaban etiquetados como Raja spp., excepto uno que estaba etiquetado como R. clavata. Los resultados mostraron que todos los productos estaban correctamente etiquetados y, además, que las especies más comunes utilizadas en los productos elaborados a partir de raya pertenecen al género Bathyraja, como B. brachyurops (35%) seguido de Amblyraja radiata (25%) y otras especies menos comunes pertenecientes a los géneros Bathyraja, Raja y Leucoraja.
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Identificación genética de gádidos: Este sistema se aplicó a 25 muestras comerciales etiquetadas como “Bacalao”. El 88% de las muestras analizadas estaban etiquetadas correctamente de acuerdo con las especies declaradas, mientras el 8% restante (2 muestras) no lo estaban (la especie indicada en la etiqueta no estaba de acuerdo la contenida). Además, se detectaron mezclas de especies en el 8% de los productos comerciales (2 muestras), pero éstos estaban etiquetados correctamente como "Gadus spp.", sin especificar las especies contenidas.
Los resultados obtenidos en estos estudios resaltan el correcto funcionamiento de las técnicas propuestas en la presente tesis doctoral, que serán de gran utilidad para la prevención de fraudes alimentarios relacionados con la seguridad y el correcto etiquetado de las especies estudiadas, garantizando la información veraz en el etiquetado de los productos a su disposición. La aplicación de estas herramientas de control se traducirá en un incremento del valor añadido de los productos elaborados, y contribuirá a hacer más competitivo y transparente el sector productor y transformador de productos alimentarios.
Las metodologías moleculares desarrolladas permitirán a las autoridades competentes realizar un control normativo de productos transformados, mediante la comprobación de los documentos que acompañan a los productos y su situación normativa en materia de seguridad, trazabilidad, verificación de la autenticidad, certificación y etiquetado, con el fin de asegurar la calidad final del producto que llega al consumidor.
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CONCLUSIONES
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CONCLUSIONES
Los métodos desarrollados en la presente tesis doctoral para la identificación de especies de carnes y pescados, basados en la técnica FINS, es decir, secuenciación de ADN y posterior análisis filogenético de las secuencias, funcionan correctamente en todo tipo de productos, tanto frescos como procesados, independientemente del grado de transformación al que hayan sido sometidos.
La utilización de marcadores mitocondriales para el desarrollo de los métodos incluidos en la presente tesis doctoral, resalta las numerosas ventajas de éstos respecto a los nucleares. Entre ellas: mayor tasa de mutación, suficientemente alta como para diferenciar especies estrechamente relacionadas pero no tan elevada como para acumular una alta variación intraespecífica que impida diseñar cebadores adecuados para grupos taxonómicos amplios; alto número de copias por célula, que permite su recuperación y utilización en casos en los que el ADN nuclear es viable; vía de transmisión materna que, al carecer de recombinación, se comporta como un bloque de genes ligados que se transmite intacto a lo largo de generaciones sucesivas. De este modo, se han podido diseñar herramientas moleculares basadas en el análisis de un fragmento del gen mitocondrial citocromo b (cytb) para diferentes especies de carnes, jureles, sardinas, anguilas y gádidos; y basados en el gen citocromo oxidasa subunidad I (COI) para diferentes especies de rayas.
Cabe destacar que estos trabajos son los más completos desarrollados hasta la fecha en cuanto al número de especies abordadas (42 especies de carnes, 25 especies de jureles, 21 especies de sardinas, 42 especies de rayas, 12 especies de anguilas y 17 especies de gádidos).
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En cuanto a las técnicas de identificación de especies de jureles, sardinas, anguilas y gádidos, su principal ventaja en relación a los trabajos previos ([57, 58, 78, 79] ; [55, 56]; [80-82]; y [83, 84], respectivamente) es que, (además de incluir un elevado número de especies), se basan en el uso de un fragmento de ADN de bajo tamaño, 239 pb en jureles y anguilas, 145 pb en sardinas y 263 pb en gádidos . Por ello, pueden ser aplicados a cualquier tipo de producto, independientemente del grado de transformación. Esto es de vital importancia, ya que el ADN se fragmenta cuando se ve sometido a altas temperaturas y presiones, procesos que son habituales en la industria alimentaria [6].
Es también destacable que la técnica desarrollada para sardinas y especies afines permite detectar mezclas de Sardina pilchardus y Sardinella aurita con un límite de detección de 5% (p/p). Esto es importante ya que permite detectar las sustituciones de estas especies de forma fraudulenta.
Así mismo, en el caso del trabajo de gádidos, su principal novedad radica en que es el único que permite la detección de mezclas de varias especies del género Gadus, concretamente G. morhua, G. macrocephalus y G. ogac, con un límite de detección de 5% (p/p). Es destacable esta característica metodológica debido a que, entre los principales formatos de comercialización del bacalao, además de entero, fileteado, precocinado… es muy usual encontrar las “Migas de bacalao” que, debido a su presentación comercial, son susceptibles de sufrir adulteración con mezclas de estas especies, ya bien sea de forma accidental o intencionada.
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En todos los trabajos que componen la presente tesis doctoral se realizó la de validación metodológica, que confirma la validez, robustez y fiabilidad de los métodos desarrollados. Para ello, se simularon los principales procesos industriales de transformación de las especies estudiadas. Es este punto es destacable la elección del marcador mitocondrial, ya que, su abundancia relativa en comparación con el ADN nuclear y su estructura circular le otorgan una mayor resistencia a la degradación inducida por el calor [85]. Por otro lado se analizaron también las diferentes salsas y especias que acompañan a muchos de estos productos, y que pueden disminuir la cantidad y calidad del ADN extraído, así como atenuar o inhibir la amplificación del ADN en la PCR [13]. De esta forma, se comprobó que las técnicas desarrolladas son aplicables a cualquier tipo de producto procesado y que el proceso de transformación no influye en la correcta identificación de las especies contenidas en los productos.
Además, todos los trabajos que componen la presente tesis doctoral exceptuando el de anguilas, incluyen una sección de aplicación a muestras comerciales, en la que se han analizado los principales formatos de comercialización de cada grupo taxonómico. En el caso de jureles, se han analizado 15 conservas y en rayas 20 muestras. Los resultados indican que todas las muestras estaban correctamente etiquetadas. En el caso de carnes, sardinas y gádidos, se han analizado 20, 83 y 25 productos respectivamente, obteniendo el 15%, 15,66% y 8% de muestras etiquetadas incorrectamente. Es destacable que estos estudios han confirmado el correcto funcionamiento de las técnicas propuestas.
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Por todo ello, se puede concluir que los métodos desarrollados son altamente específicos, fiables reproducibles, rápidos, simples y eficaces. Entre las principales ventajas de estos sistemas cabe destacar que los resultados no se ven afectados por la variabilidad intraespecífica.
Es también destacable que todos ellos incluyen un elevado número de especies estudiadas dentro de cada metodología. Esto es importante dado que, aunque algunas ya han sido incluidas individualmente o en pequeños grupos en otros estudios, no existe hasta la fecha ningún trabajo que englobe tantas especies, facilitando el análisis e identificación de muestras desconocidas dentro de cada grupo taxonómico.
Sus posibles aplicaciones son:
Control normativo de productos crudos y procesados. Autentificación de especies importadas. Verificación de la trazabilidad de diferentes lotes de pesca a lo largo de la cadena comercial. Control del correcto etiquetado. Protección de los derechos del consumidor, quien dispondrá de mayor información sobre los productos que consume. Competencia equitativa entre los operadores pesqueros. Control de pesquerías.
Por tanto, todas estas metodologías pueden ser utilizadas como métodos de rutina en diferentes laboratorios de control de calidad, autoridades competentes… para la detección de fraudes intencionados o accidentales en productos alimentarios y así como para evaluar su correcta trazabilidad.
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