PCR-SSCP: A Simple Method for the Authentication of Grouper (Epinephelus guaza), Wreck Fish (Polyprion americanus), and Nile Perch (Lates niloticus) Fillets Luis Asensio, Isabel Gonza ´ lez,* Alicia Ferna ´ ndez, Miguel A. Rodrı ´guez, Pablo E. Herna ´ ndez, Teresa Garcı ´a, and Rosario Martı ´n Departamento de Nutricio ´n y Bromatologı ´a III (Higiene y Tecnologı ´a de los Alimentos), Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain A method of DNA analysis has been developed to verify the authenticity of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. A short fragment (208 bp) of the mitochondrial 12S rRNA gene was amplified by the polymerase chain reaction and analyzed by single-strand conformation polymorphism to get species-specific patterns of single- stranded DNA (ssDNA). DNA strands were separated by native polyacrylamide gel electrophoresis and visualized by silver staining. Discrimination among the three fish species studied was possible, because each one expressed a specific ssDNA pattern. Keywords: Fish species identification; PCR-SSCP; 12S rRNA gene; Epinephelus guaza; Polyprion americanus; Lates niloticus INTRODUCTION Identification of fish species in the marketplace can be uncertain whenever the usual external characteris- tics such as skin pigmentation, shape, size, and appear- ance are removed on processing and only a portion of flesh is available. For this reason, when the whole fish is transformed into fillets, opportunities for substitution or adulteration increase (1). Nile perch (Lates niloticus) fillets are frequently labeled and marketed either as grouper (Epinephelus guaza) or as wreck fish (Polyprion americanus) because of the higher popularity and quality of the two latter species. Additionally, grouper and wreck fish are closely related species (family Serranidae) that may be mis- identified in the market and are commonly sold as grouper, which is more demanded by consumers. The development of analytical methods for fish species identification is therefore necessary for preventing willful as well as unintentional substitution of different fish species (2). In recent years, various protein-based techniques including immunological, electrophoretic, and chromato- graphic methods have been used for fish species iden- tification. Among these, immunological techniques have offered an alternative means for the identification of fish species and proved to be suitable for routine analysis of a large number of samples (3-5). Electrophoretic techniques and methods such as liquid chromatography and high-performance liquid chromatography (HPLC) have also been reported, allowing identification of processed fish products (6-9). However, these methods are laborious and require substantial equipment, and a large data bank of various seafood species is needed for effective protein profile comparison. Advances in DNA technology have led to rapid development of genetic methods for fish species identi- fication. DNA offers advantages over proteins, including stability at high temperature, presence in all tissue types, and greater variation with genetic code (1). DNA can be analyzed using techniques such as sequencing, DNA-DNA hybridization, and the polymerase chain reaction (PCR), which are based on the detection of species-specific DNA sequences in food products. In particular, PCR-based techniques have a high potential because of their rapidity, increased sensitivity, and specificity (10). PCR-based methods commonly used for fish species identification include random amplified polymorphic DNA (RAPD) (11, 12), PCR restriction fragment length polymorphism (PCR-RFLP) (13-15), and PCR single- strand conformational polymorphism (PCR-SSCP) (16-18). RAPD, also known as arbitrary primed PCR (AP- PCR), is a very fast method that provides a great number of polymorphisms, but its major drawback is poor reproducibility of the results. PCR-RFLP has quickly gained acceptance among fish species identifica- tion techniques. However, this technique detects differ- ences in DNA sequences only when the differences are present in the specific recognition site for the corre- sponding endonuclease. This limitation has been over- come by the PCR-SSCP technique, which is based on the relationship between the electrophoretic mobility of a single-stranded DNA (ssDNA) and its folded confor- mation, which in turn reflects the nucleotide sequence (19). In the PCR-SSCP technique, the amplified product is denatured to a single-stranded form and electro- phoresed on a non-denaturing polyacrylamide gel. Any difference in the sequences causes a shift in the mobility of the analyzed molecule, which is visualized at the end of the process (20). In our study, amplification of a short fragment of the mitochondrial 12S rRNA gene by PCR, followed by SSCP analysis of the amplicons, was used for the * Corresponding author (telephone 34-913943751; fax 34- 913943743; e--mail gonzalzi@eucmax.sim.ucm.es). 1720 J. Agric. Food Chem. 2001, 49, 1720-1723 10.1021/jf001185w CCC: $20.00 © 2001 American Chemical Society Published on Web 03/06/2001