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.

Molecular identification of Atlantic goliath grouper Epinephelus itajara (Lichtenstein, 1822) (Perciformes: Epinephelidae) and related commercial species applying multiplex PCR

“…Molecular markers used in the multiplex fingerprinting technique for the authentication, control, and inspection of fishery stocks are based on the design of specific oligonucleotides and markers derived from the sequencebased amplification of the 5S, 12S, 18S (Asensio, 2008, Asensio et al, 2009, 16S (Ulrich et al, 2013), Cyt b (Melo Palmeira et al, 2013), and COI genes , as well as fragment length polymorphisms, RFLPs (Paine et al 2007;Torres et al 2013) or even nuclear markers, such as ITS 2 (Feldheim et al, 2010), SNPs (Torres et al, 2013) and microsatellites (Rodrigues et al, 2011), and realtime PCR techniques (Trotta et al, 2005). The COI gene is a highly effective and reliable tool for the diagnosis of the authenticity of fishery products (Veneza et al, 2014), and the multiplex PCR technique has proven especially effective for the detection of endangered species De-Franco et al, 2010;Alexandre de-Franco et al, 2012).…”

“…Carvalho et al (2015) has shown the DNA barcoding to be an effective molecular tool for the detection of commercially-valuable species such as the groupers, while Torres et al (2013) successfully applied PCR RFLP and SNP detection techniques to the identification of specimens of E. itajara, E. morio, M. bonaci, and M. marginata. The multiplex PCR technique, based on the COI gene Mendonça et al, 2010) and other mitochondrial (Trotta et al, 2005;Hashimoto et al, 2010;Ravago-Gotanco et al, 2010) and nuclear genes (Shivji et al, 2002;Chapman et al, 2003;Rodrigues et al, 2011), has proven to be an efficient and rapid low-cost tool that is safe and easy to apply (Abercrombie et al, 2005). This technique permits the identification of illegal fishing by the authorities, even when the catches have been processed for consumption, and contribute to conservation practices on a worldwide scale .…”

Identifikacja Gatunku Karp Zwyczajny (Cyprinus Carpio) Przy Użyciu Real-Time PCR

“…Thus, for instance, Sotelo et al [26] used the TaqMan assay for the identification and quantification of Cod (Gadus morhua). Trotta et al [30] used the Real-Time PCR for the identification of fish fillets from Grouper (Genera Epinephelus and Mycteroperca) and common substitute species. Hird et al [5] The development of analytical methods for fish species identification may help to detect and avoid willful, as well as unintentional substitution of different fish species and thus enforce labeling regulations [2,12,15,29].…”

The Barcode of Life Initiative: synopsis and prospective societal impacts of DNA barcoding of Fish

“…86 Such a tool could also be used for detection of fraudulent species substitutions in fish markets and fish food products, a practice that is generating concern among consumers. 87 A striking example comes from the Red Snapper (Lutjanus campechanus), which is one of the most economically important fisheries in the Gulf of Mexico, and which has been subject to stringent fishing restrictions due to stock depletion. Marko and colleagues 88 used sequences of the mtDNA gene cytochrome b, in an approach very similar to DNA barcoding, to show that as much as 77% of the L. campechanus fillets were mislabelled in USA markets.…”