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Dwiyitno D, Hoffman S, Parmentier K, Keer CV. 2021. Universal primer design for crustacean and bivalve-mollusc authenticity based on cytochrome-b gene. Biodiversitas 23: 17-24. Fish and seafood authenticity is important to support traceability practices and protect the public from economic fraud and adulteration. Molecular-based techniques of PCR a...
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... A PCR assay based on the use of species-specific primers annealing on mitochondrial DNA (mtDNA) could meet these three requirements. Several mitochondrial genes have provided useful PCRbased molecular markers to differentiate bivalve species (e.g., Ardura et al. 2015;Nantón et al. 2015;Dwiyitno et al. 2022), but in the case of cockles, only whole-molecule RFLPs have been described (Brock and Christiansen 1989). Therefore, the aim of this work was to develop a simple and fast method consisting of a new PCR assay based on the mitochondrial cytochrome c oxidase subunit I (COI) gene to diversify the options for molecular differentiation of the two species of cockles and to assist in conservation and sustainable management efforts. ...
Cerastoderma edule and C. glaucum are two species of cockles that co-exist in European waters. They are morphologically similar but exhibit remarkable differences in biological, ecological, and genetic aspects, as well as in resistance to parasites (e.g., Martellia cochilia) and in disease incidence (e.g., disseminated neoplasia). Moreover, they differ in their economic significance; while C. edule represents a highly valuable marine resource, C. glaucum is only marginally fished. The aim of this work was to develop a simple and fast method that, for the first time, uses the sequence of a mitochondrial gene for the molecular differentiation of the two cockle species. A total of 304 partial sequences of the cytochrome c oxidase subunit I (COI) gene, retrieved from the Nucleotide database, were used to design two sets of species-specific primers to generate PCR products of different sizes (322 bp in C. glaucum and 247 bp in C. edule). The discriminatory ability of the PCR assay was tested in cockles from the Spanish, French, and Italian coasts with successful differentiation in all cases. This novel molecular identification method requires minimal technical equipment and can be carried out in one working day. For its simplicity, it can be very useful for conservation and sustainable management of the two cockle species, facilitating the assessment of distribution, abundance and relative sensitivity to viruses, parasites and diseases.
... As demonstrated in the alignment result, this gene showed a good conservative sequence between different species. This was in accordance with the other works that have successfully applied this gene to identify fish species in processed products [23,24]. ...
... For example, cytb gene was effectively employed for the development of multiplex PCR used for the detection of five species of codfish [25]. Another study also reported that the primers designed based on this gene potentially identified the species of crustacean and mollusk samples [24]. Moreover, Fernandes et al. (2017) [17] reported the success of using cytb gene to differentiate four types of gadoid fish ...
Overall quality attributes between deskinned Asian seabass and mangrove red snapper fillet are similar. This may lead to the substitution of Asian seabass, a cheaper species, with the latter. Therefore, the authentication method for distinguishing these two species could conquer adulteration. Four primers were designed, based on mitochondrial cytb gene alignment. Polymerase chain reaction (PCR) was performed using the DNA of both species with 35 cycles of amplification. The primers provided the amplicons with the size of 480, 396, 185, and 268 for primers 1, 2, 3, and 4, respectively. All primers successfully differentiated Asian seabass from mangrove red snapper both raw and cooked fillets from at least 30 mg of the sample weight. Moreover, primer 4 showed the highest specificity when tested with other 5 species of fish generally sold in the Thai market. The limit of detection was around 6 ng of Asian seabass DNA in a total of 120 ng mangrove red snapper DNA. Therefore, the PCR-based method using primer 4 was a useful tool for the identification of Asian seabass adulterated in mangrove red snapper fillets or slices. This primer could be implemented for authentication of fish and fish products to prevent mislabeling for customers.
