title: Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species.
abstract: Mitochondrial 16S rRNA sequences from morphological validated grouper (Epinephelus aeneus, E. caninus, E. costae, and E. marginatus; Mycteroperca fusca and M. rubra), Nile perch (Lates niloticus), and wreck fish (Polyprion americanus) were used to develop an analytical system for group diagnosis based on two alternative Polymerase Chain Reaction (PCR) approaches. The first includes conventional multiplex PCR in which electrophoretic migration of different sizes of bands allowed identification of the fish species. The second approach, involving real-time PCR, produced a single amplicon from each species that showed different Tm values allowing the fish groups to be directly identified. Real-time PCR allows the quick differential diagnosis of the three groups of species and high-throughput screening of multiple samples. Neither PCR system cross-reacted with DNA samples from 41 common marketed fish species, thus conforming to standards for species validation. The use of these two PCR-based methods makes it now possible to discriminate grouper from substitute fish species.
authors.affiliation: Departamento de Bioquímica y Biología Molecular IV, Facultad Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Michele
authors.initials: M
authors.lastname: Trotta
authors.forename: Susana
authors.initials: S
authors.lastname: Schönhuth
authors.forename: Tiziana
authors.initials: T
authors.lastname: Pepe
authors.forename: M Luisa
authors.initials: ML
authors.lastname: Cortesi
authors.forename: Antonio
authors.initials: A
authors.lastname: Puyet
authors.forename: José M
authors.initials: JM
authors.lastname: Bautista
mesh_terms: D000818:Animals; D004247:DNA / Q000032:analysis; D005396:Fish Products / Q000145:classification*; D005399:Fishes / Q000145:classification* / Q000235:genetics*; D010473:Perciformes / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004247:DNA