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Universidad Autónoma De Nuevo León Facultad De Ciencias Biológicas

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Universidad Autónoma De Nuevo León Facultad De Ciencias Biológicas UNIVERSIDAD AUTÓNOMA DE NUEVO LEÓN FACULTAD DE CIENCIAS BIOLÓGICAS TESIS MODELO ECOLÓGICO PARA REDUCIR LA CARGA DE AGENTES ENTEROPATÓGENOS MICROBIANOS PRESENTES EN EL PARQUE NACIONAL CASCADA DE BASSASEACHIC, EN OCAMPO, CHIHUAHUA POR MARÍA CARMEN ELIZABETH DELGADO GARDEA COMO REQUISITO PARCIAL PARA OBTENER EL GRADO DE DOCTOR EN CIENCIAS CON ACENTUACIÓN EN MICROBIOLOGÍA J U N I O, 2 0 1 6 UNIVERSIDAD AUTÓNOMA DE NUEVO LEÓN FACULTAD DE CIENCIAS BIOLÓGICAS SUBDIRECCIÓN DE ESTUDIOS DE POSGRADO TESIS MODELO ECOLÓGICO PARA REDUCIR LA CARGA DE AGENTES ENTEROPATÓGENOS MICROBIANOS PRESENTES EN EL PARQUE NACIONAL CASCADA DE BASSASEACHIC, EN OCAMPO, CHIHUAHUA POR MARÍA CARMEN ELIZABETH DELGADO GARDEA COMO REQUISITO PARCIAL PARA OBTENER EL GRADO DE DOCTOR EN CIENCIAS CON ACENTUACIÓN EN MICROBIOLOGÍA J U N I O, 2 0 1 6 MODELO ECOLÓGICO PARA REDUCIR LA CARGA DE AGENTES ENTEROPATÓGENOS MICROBIANOS PRESENTES EN EL PARQUE NACIONAL CASCADA DE BASSASEACHIC, EN OCAMPO, CHIHUAHUA Comité de Tesis __________________________________________________________ Dra. Patricia Tamez Guerra Presidente __________________________________________________________ Dr. Ricardo A. Gómez Flores Secretario __________________________________________________________ Dr. José Alberto Valadez Lira Vocal 1 __________________________________________________________ Dra. Laura Trejo Ávila Vocal 2 __________________________________________________________ Dr. Juan Francisco Contreras Cordero Vocal 3 MODELO ECOLÓGICO PARA REDUCIR LA CARGA DE AGENTES ENTEROPATÓGENOS MICROBIANOS PRESENTES EN EL PARQUE NACIONAL CASCADA DE BASSASEACHIC, EN OCAMPO, CHIHUAHUA Dirección de Tesis __________________________________________________________ Dra. Patricia Tamez Guerra Directora __________________________________________________________ Dra. Rocío Infante Ramírez Directora externa AGRADECIMIENTOS El presente proyecto fue realizado en las instalaciones del Laboratorio de Inmunología y Virología, en la Universidad Autónoma de Nuevo León, México, Facultad de Ciencias Biológicas (DEMI-FCB-UANL), y por la beca CONACyT (beca a MCEDG y el proyecto CB2010/155771 para PTG). En colaboración con la Universidad Autónoma de Chihuahua, Facultad de Ciencias Químicas y la Comisión Nacional de Áreas Naturales Protegidas (CONANP) proyecto UACH-CONANP (CONANP/DR03/08/PN01/PROCODES/1259/13) to RIR. DEDICATORIA ÍNDICE 1 INTRODUCCIÓN........................................................................................................................ 1 2 ANTECEDENTES....................................................................................................................... 3 2.1 Parque Nacional Cascada de Bassaseachic................................................................................. 3 2.2 El agua como recurso no renovable............................................................................................ 3 2.3 Fuentes de abastecimiento de agua potable. .............................................................................. 4 2.4 Importancia de la Calidad de agua. .............................................................. ........................... 5 2.5 Bacterias contaminantes del agua. ............................................................... ............................. 6 2.6 Virus contaminantes en agua..................................................................................................... 10 2.7 Determinación de microorganismos en agua.............................................................................. 14 2.8 Métodos de concentración de patógenos en agua.................................... ................................. 16 2.9 Métodos de Potabilización de agua............................................................ .............................. 18 3 HIPÓTESIS...................................................................................................... ........................... 21 4 OBJETIVO GENERAL.................................................................................. .......................... 22 5 OBJETIVOS PARTICULARES.................................................................... .......................... 22 6 ESTRATEGIA EXPERIMENTAL................................................................ .......................... 23 7 METODOLOGÍA............................................................................................ ........................... 24 7. Muestreo..................................................................................................... ................................ 24 7.1Análisis Físico-Químico................................................................................ .......................... 24 7.1.1 pH (NMX-AA-008-SCFI-2011)...................................................................................... 24 7.1.2 Conductividad eléctrica (NMX-AA-093-SCFI-2000)............................... .................... 24 7.1.3 Cloruros (NMX-AA-073-SCFI-2001)........................................................ .................... 25 7.1.4 Dureza total (NMX-AA-072-SCFI-2001)....................................................................... 25 7.1.5 Alcalinidad (NMX-AA-036-SCFI-2001 12)................................................................... 26 7.1.6 Acidez (NMX-AA-036-SCFI-2001 12)...................................................... .................... 27 7.2 Conteo de coliformes fecales y totales (PROY-NMX-AA-042-SCFI-2005)... ....................... 27 7.3 Análisis estadístico para conteo de coliformes fecales y totales, por punto de muestreo y época del año. .......................................................................................................................... 28 7.4 Aislamiento de bacterias enteropatógenas. ................................................................................ 28 7.5 Identificación bioquímica de bacterias enteropatógenas. ......................................................... 30 7.6 Técnica de asborción-elución de virus (VIRADEL)................................................................. 30 7.7 Muestras control para captura de virus...................................................................................... 31 7.8 Preparación de controles internos virales.................................................. ............................... 31 7.8.1 Preparación del inserto a clonar................................................... ................................. 32 7.8.2 Reacción de ligación. .................................................................... .............................. 32 7.8.3 Transformación de células competentes JV109. .............................. .............................. 33 7.9 Extracción de RNA por el método de TRIZOL. ......................................... ............................ 33 7.10 Determinación de RV por RT-PCR punto final......................................... ............................ 34 7.10.1 Condiciones de RT para detección de RV............................................ ......................... 34 7.10.2 Condiciones para la PCR para detección de RV........................................................... 35 7.11 Determinación de NV por RT-PCR punto final........................................................................ 36 7.11.1 Condiciones de RT para detección de NV................................. ................................ 36 7.11.2 Condiciones para la PCR para detección de NV.......................... ................................ 37 7.12 Electroforesis en gel de agarosa al 1%.................................................... ................................ 38 7.13 Preparación de la Curva de calibración para qPCR de NV y RV............................................. 38 7.14 Determinación de RV por RT-qPCR SYBR ® Green.................................. .......................... 41 7.14.1 Condiciones de qPCR SYBR ® Green. Detección de RV........................................... 41 7.15 Determinación de NV por RT-qPCR SYBR ® Green.............................................................. 43 7.15.1 Condiciones de qPCR SYBR ® Green. Detección de NV............................................ 43 7.16 Análisis filogenético de muestras positivas............................................................................. 44 7.17 Diseño del prototipo del generador de Cloro............................................................................ 45 7.18 Determinación de Cloro disponible........................................................ ................................. 46 7.19 Estandarización del prototipo del generador de cloro............................. ................................. 46 7.20 Análisis estadístico de los resultados del generador de cloro. ................................................. 47 7.21 Diseño del prototipo de membranas de UF............................................ ................................. 47 7.22 Método de prueba para evaluar la eficiencia en reducción bacteriana..................................... 48 7.22.1 Preparación de cultivos y sub-cultivos de referencia................................................... 48 7.22.2 Preparación del agua de prueba.................................................................................... 48 7.22.3 Evaluación de la reducción bacteriana........................................................................... 49 7.23 Determinación de mesófilos aerobios (NOM-092-SSA1-1994).............................................
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