Identification of Nile Perch (<I>Lates Niloticus</I>), Grouper

1248

Journal of Food Protection, Vol. 63, No. 9, 2000, Pages 1248±1252 Copyright ᮊ, International Association for Food Protection

Identi®cation of Nile Perch (Lates niloticus), Grouper (Epinephelus guaza), and Wreck Fish (Polyprion americanus)by Polymerase Chain Reaction±Restriction Fragment Length Polymorphism of a 12S rRNA Gene Fragment

LUIS ASENSIO, ISABEL GONZAÂ LEZ,* ALICIA FERNAÂ NDEZ, ANA CEÂ SPEDES, PABLO E. HERNAÂ NDEZ, TERESA GARCIÂA, AND ROSARIO MARTIÂN Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/9/1248/1588823/0362-028x-63_9_1248.pdf by guest on 27 September 2021 Departamento de NutricioÂn y BromatologõÂa III (Higiene y TecnologõÂa de los Alimentos), Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain

MS 99-328: Received 2 November 1999/Accepted 4 January 2000

ABSTRACT

Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the 12S rRNA gene has been used for the speci®c identi®cation of Nile perch (Lates niloticus), grouper (Epinephelus guaza), and wreck ®sh (Polyprion americanus). Ampli®cation of DNA isolated from muscle samples was carried out using a set of primers ¯anking a region of 436 bp from the mitochondrial 12S rRNA gene. Digestions of the PCR products with RsaI and Sau96I endonucleases, followed by agarose gel electrophoresis of the digested PCR products, yielded speci®c pro®les that enabled direct identi®cation of each species analyzed.

Knowledge of the species of ®sh destined for human analysis of proteins, many of which are heat labile. As an consumption is necessary for both nutritional and economic alternative to protein analysis, a number of DNA-based reasons, including the adherence to fair pricing policies and identi®cation techniques have been developed. The DNA the meeting of quality control requirements and legal reg- molecule offers a number of advantages when compared to ulations. However, the identi®cation of ®sh species be- proteins: all cell types of an individual contain identical comes a problem when the morphological characters are genetic information independent of the origin of the sample, removed on processing and only a portion of ¯esh is avail- the DNA molecule is stable at high temperature, and the able (12). Consequently, there is a risk of willful or unin- information content of DNA is greater than that of proteins tentional substitution of lower valued ®sh species for high (5). valued ®sh in seafood products. In recent years, the polymerase chain reaction (PCR), Nile perch (Lates niloticus) ®llets are many times la- followed by nucleotide sequencing or restriction fragment beled and marketed either as grouper (Epinephelus guaza) length polymorphism (RFLP) analysis, has permitted the or as wreck ®sh (Polyprion americanus). Although the identi®cation of different ®sh species (6, 8, 16). According three ®sh species ®llets are similar in texture, the quality to this, we report in this paper a method for the speci®c attributes and price are higher for the two latter ones. Ad- identi®cation of Nile perch (L. niloticus), grouper (E. gua- ditionally, grouper and wreck ®sh are closely related spe- za), and wreck ®sh (P. americanus) based on PCR-RFLP cies that may be misidenti®ed in the marketplace and are analysis of a conserved region in the mitochondrial 12S commonly sold as grouper, which is more in demand by rRNA gene. This method can be applied to the detection consumers. The development of suitable analytical methods of fraudulent or unintentional mislabeling of these species for ®sh species identi®cation are therefore of great interest in the processed product market. to enforce labeling regulations in the authentication of ®sh species (9, 11). MATERIALS AND METHODS Over the last few years, several molecular methods Sample selection and DNA extraction. Nile perch, grouper, have been developed for ®sh species identi®cation, includ- and wreck ®sh samples were obtained from Mercamadrid (Ma- ing electrophoretic, chromatographic, and immunological drid, Spain) and other local markets. Every specimen was mor- techniques (1, 7, 10, 18, 20). Although most of these meth- phologically identi®ed following the keys of Bauchot and Pras ods are of considerable value in certain instances, they are (3). Total cellular DNA was extracted from ®sh muscle according not convenient for routine sample analysis because they are to a previously described procedure (8). Fifteen individuals of relatively costly, time consuming, and complex to perform. each species were analyzed. Moreover, they have the disadvantage of relying on the PCR ampli®cation of a 436-bp fragment from the 12S * Author for correspondence. Tel: 34-913943751; Fax: 34-913943743; rRNA gene. The set of primers used for PCR ampli®cation of the E-mail: [email protected]. 12S rRNA gene were 12S1 and 12S2 oligonucleotides (17): J. Food Prot., Vol. 63, No. 9 PCR-RFLP IDENTIFICATION OF FISH SPECIES 1249

12S1: 5Ј-AAACTAGGATTAGATACCCTATTAT-3Ј (25mer) 12S2: 5Ј-AAGAGCGACGGGCGATGTGT-3Ј (20mer) Double-stranded ampli®cations were carried out in a ®nal volume of 50 ␮l, containing 10 mM Tris-HCl, pH 8.8, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 0.2 mM each of dATP, dTTP, dGTP, and dCTP, 50 pmol of each primer, 750 ng of tem- plate DNA, and 2 U of DynaZyme II DNA polymerase (Finnzy- mes Oy, Espoo, Finland). PCR was carried out in a Progene ther- mal cycler (Techne Ltd., Cambridge, UK) programmed to perform a denaturation step of 93ЊC for 2 min, followed by 35 cycles consisting of 30 s at 93ЊC,30sat50ЊC, and 45 s at 72ЊC. The last extension step was 5 min longer.

Electrophoresis of a 10-␮l portion of the ampli®cation prod- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/9/1248/1588823/0362-028x-63_9_1248.pdf by guest on 27 September 2021 uct was performed for 45 min at 100 V in a 1.5% D1 (Hispanlab S.A., Alcobendas, Spain) agarose gel, containing ethidium bromide (1 ␮g/ml) in Tris-acetate buffer (0.004 M Tris-acetate, 0.001 M EDTA, pH 8.0). DNA fragments were visualized by UV trans- illumination and analyzed using a Geldoc 1000 UV Fluorescent Gel Documentation System±PC (Bio-Rad Laboratories, Hercules, FIGURE 1. Electrophoretic analysis of the PCR products from Ga.). the 12S rRNA gene. M ϭ 1 kb plus DNA ladder. Samples in lanes are as follows: 1, wreck ®sh (P. americanus); 2, grouper (E. Cleanup and sequencing of the PCR products. Electro- guaza); 3, Nile perch (L. niloticus); and 4, negative control. An phoresis of 80 ␮l of PCR products from each species was per- arrow indicates the 436-bp fragment. formed for 45 min at 100 V in a 1% LM2 (Hispanlab) agarose gel containing ethidium bromide (1 ␮g/ml) in Tris-acetate buffer. Each DNA fragment was excised from the agarose gel using a quences, two restriction endonucleases, RsaI and Sau96I, sterile scalpel. The gel slice was puri®ed using the Qiaquick gel were found to be potentially useful for the speci®c identi- extraction kit (Qiagen GmbH, Hilden, Germany), according to the ®cation of Nile perch, grouper, and wreck ®sh (Fig. 2). manufacturer's instructions. The DNA was eluted in 20 ␮l of sterile distilled water. The concentration of the puri®ed PCR product Results following digestion of the PCR products from was estimated by agarose gel electrophoresis using a standard the three species analyzed showed that band sizes obtained (Mass Ruler; Bio-Rad) as reference marker. A Geldoc 1000 Sys- by electrophoresis in 3.5% MS8 agarose gel were in agree- tem±PC (Bio-Rad) was used for that purpose. ment with the expected sizes for the restriction fragments Puri®ed PCR products were sequenced at the Centro de In- inferred from the sequence analysis: RsaI cleaved the an- vestigaciones BioloÂgicas, Consejo Superior de Investigaciones alyzed 12S rRNA segment of wreck ®sh into two fragments CientõÂ®cas (Madrid, Spain). DNA sequencing was accomplished of 346 and 90 bp; grouper into three fragments of 189, 173, using dRhodamine Terminator Cycle Sequencing Ready Reaction and 74 bp; and Nile perch into two fragments of 234 and Kit (Perkin-Elmer Applied Biosystems Division, Foster City, Ca- 202 bp (Fig. 3). Similarly, digestions perfomed with Sau96I lif.) in an ABI Prism model 377 DNA sequencer (Perkin-Elmer). resulted in two DNA fragments of 402 and 34 bp in wreck Direct sequences of each PCR fragment were obtained for three ®sh PCR products; three fragments of 224, 178, and 34 bp samples of each Nile perch, grouper, and wreck ®sh species. were obtained in grouper; and, ®nally, the two Sau96I re- Restriction site mapping and enzymatic digestion of PCR striction sites present in Nile perch sequences yielded two products. Restriction maps of 12S rRNA gene sequences from DNA fragments of 224 and 212 pb (Fig. 4). Results ob- Nile perch, grouper, and wreck ®sh were obtained using the Wis- tained with 15 different individuals from each species did consin Package, version 9.0. Two endonucleases were then se- not show intraspeci®c polymorphism for the restriction sites lected for restriction analysis of the ampli®ed PCR products: RsaI tested. and Sau96I (New England Biolabs, Beverly, Mass.). Digests were performed in 20 ␮l volumes with 100 ng ampli®ed DNA, 10 U DISCUSSION enzyme, and a 1 to 10 dilution of the manufacturer's recommend- ed 10ϫ digestion buffer. Digestions were incubated for 15 h at In view of the increases in international seafood trade 37ЊC. The resulting fragments were separated by electrophoresis and the existing laws requiring that products be correctly in a 3.5% MS8 agarose gel (Hispanlab) containing 1 ␮g/ml eth- labeled, there is a growing need for techniques that enable idium bromide for1hat80V.Thesizes of the DNA fragments authentication of ®sh and seafood products (13). In search were estimated by comparison with a commercial 1-kb plus DNA for fast and simple genetic techniques, PCR-RFLP has ladder (Gibco BRL). gained acceptance among ®sh species identi®cation methods, because it is much easier to perform and less costly RESULTS than conventional DNA sequencing and nucleotide se- Primers 12S1 and 12S2 consistently ampli®ed a frag- quence analysis (14). In this work we have developed a ment of 436 bp from the 12S rRNA gene of Nile perch, method for the speci®c discrimination between Nile perch, grouper, and wreck ®sh (Fig. 1). PCR products from at least grouper, and wreck ®sh based on RFLP analysis of PCR- three individuals of each species were sequenced. After de- ampli®ed products from the mitochondrial 12S rRNA gene. tailed comparison of the restriction maps of 12S rRNA se- Mitochondrial DNA has been widely used as a marker 1250 ASENSIO ET AL. J. Food Prot., Vol. 63, No. 9 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/9/1248/1588823/0362-028x-63_9_1248.pdf by guest on 27 September 2021 J. Food Prot., Vol. 63, No. 9 PCR-RFLP IDENTIFICATION OF FISH SPECIES 1251 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/9/1248/1588823/0362-028x-63_9_1248.pdf by guest on 27 September 2021

FIGURE 3. Electrophoretic analysis (3.5% MS8 agarose) of the FIGURE 4. Electrophoretic analysis (3.5% MS8 agarose) of the restriction fragments obtained from PCR products digested with restriction fragments obtained from PCR products digested with RsaI endonuclease. Samples are as follows: 1, undigested PCR Sau96I endonuclease. Samples are as follows: 1, undigested PCR product; 2, wreck ®sh (P. americanus); 3, grouper (E. guaza); 4, product; 2, wreck ®sh (P. americanus); 3, grouper (E. guaza); 4, Nile perch (L. niloticus); M, 1 kb plus DNA ladder. An arrow Nile perch (L. niloticus); M, 1 kb plus DNA ladder. An arrow indicates the 436-bp fragment. indicates the 436-bp fragment. in species identi®cation (2, 15, 19). In particular, the mi- native techniques, such as direct sequencing of PCR prod- tochondrial encoded gene for 12S rRNA satis®es most of ucts, PCR-RFLP offers the advantages of being simpler, the requirements for the development of species differen- cheaper, and especially useful for routine ®sh species iden- tiation techniques: it has an acceptable length to permit de- ti®cation in the large-scale studies such as required in in- tection of sequence differences between congeneric species; spection programs. it accumulates point mutations quickly enough to allow, in ACKNOWLEDGMENTS most cases, the discrimination of even closely related species; and there are different sequences available in the da- This work was supported by the ComisioÂn Interministerial de Cien- cia y TecnologõÂa (CICYT, Spain) (project ALI 98-0696). We are indebted tabases. Additionally, the high copy number and the con- to AÂ ngel MendizaÂbal (S.V.O., Mercamadrid) for kindly supplying ®sh served regions of mitochondrial DNA are basic traits for samples. the high sensitivity of PCR-based methods applied to spe- REFERENCES cies identi®cation (4, 21). The mitochondrial primers 12S1 and 12S2 (17) used 1. Armstrong, S. G., and D. N. Leach. 1992. The use of HPLC protein in this work successfully ampli®ed a conserved 436-bp re- pro®les in ®sh species identi®cation. Food Chem. 44:147±155. gion from the 12S rRNA gene of all Nile perch, grouper, 2. Bartlett, S. E., and W. S. Davidson. 1991. Identi®cation of Thunnus tuna species by the polymerase chain reaction and direct sequence and wreck ®sh individuals analyzed. The GenBank and analysis of their mitochondrial cytochrome b genes. J. Fish. Aquat. EMBL databases were searched for sequences of the 12S Sci. 48:309±317. rRNA gene from these species, but no such sequences were 3. Bauchot, M. L., and A. Pras. 1993. GuõÂa de los peces de mar de available. Thus, three PCR products from each, Nile perch, EspanÄa y Europa, 3rd ed. Ediciones Omega, S. A., Barcelona, Spain. grouper, and wreck ®sh, were sequenced for subsequent re- 4. Brown, J. R., K. Beckenbach, A. T. Beckenbach, and M. J. Smith. 1996. Length variation, heteroplasmy and sequence divergence in striction map analysis and endonuclease selection. The re- the mitochondrial DNA of four species of sturgeon (Acipenser). Ge- sults obtained in this study showed that RsaI and Sau96I netics 142:525±535. endonucleases independently provided speci®c identi®ca- 5. Cano, R. J., H. N. Poinar, N. J. Pieniazek, A. Acra, and G. O. Poinar. tion of Nile perch, grouper, and wreck ®sh after PCR-RFLP 1993. Ampli®cation and sequencing of DNA from a 120±135-mil- analysis of the mitochondrial 12S rRNA fragments. lion-year-old weevil. Nature 363:536±538. 6. Carrera, E., T. GarcõÂa,A.CeÂspedes, I. GonzaÂlez, A. FernaÂndez, P. E. The PCR-RFLP method proposed in this work pro- HernaÂndez, and R. MartõÂn. 1999. Salmon and trout analysis by PCR- vides a powerful tool for discrimination between Nile RFLP for identify authentication. J. Food Sci. 64:410±413. perch, grouper, and wreck ®sh species. Compared to alter- 7. Carrera, E., T. GarcõÂa,A.CeÂspedes, I. GonzaÂlez, B. Sanz, P. E. Her-

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FIGURE 2. DNA sequences from part of the 12S rRNA gene of two Nile perch (L. niloticus), two grouper (E. guaza), and two wreck ®sh (P. americanus) samples. RsaI and Sau96I restriction sites are shown with shadow. Bold type nucleotides in the Nile perch sequence indicate the position of primers 12S1 and 12S2 used for PCR ampli®cation. 1252 ASENSIO ET AL. J. Food Prot., Vol. 63, No. 9

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