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http://dx.doi.org/10.5352/JLS.2017.27.7.746

Development of a Multiplex PCR Assay for Rapid Identification of Larimichthys polyactis, L. crocea, Atrobucca nibe, and Pseudotolithus elongates  

Noh, Eun Soo (Biotechnology Research Division, National Institute of Fisheries Science)
Lee, Mi-Nan (Biotechnology Research Division, National Institute of Fisheries Science)
Kim, Eun-Mi (Biotechnology Research Division, National Institute of Fisheries Science)
Park, Jung Youn (Biotechnology Research Division, National Institute of Fisheries Science)
Noh, Jae Koo (Biotechnology Research Division, National Institute of Fisheries Science)
An, Cheul Min (Jeju Fisheries Research Institute, National Institute of Fisheries Science)
Kang, Jung-Ha (Biotechnology Research Division, National Institute of Fisheries Science)
Publication Information
Journal of Life Science / v.27, no.7, 2017 , pp. 746-753 More about this Journal
Abstract
In order to rapidly identify four drums species, Larimichthys polyactis, L. crocea, Atrobucca nibe, and Pseudotolithus elongates, a highly efficient and quick method has been developed using multiplex polymerase chain reaction (PCR) with species-specific primers. Around 1.4 kbp of the mitochondrial COI gene sequences from the four drums species were aligned, and species-specific forward primers were designed, based on the single nucleotide polymorphism (SNP). The optimal conditions for PCR amplification were selected through cross-reactivity, using a gradient PCR method. The PCR results demonstrated species-specific amplification for each species at annealing temperatures between 50 and $62^{\circ}C$. Multiplex species-specific PCR (MSS-PCR) amplification reactions with four pairs of primers were performed for sixteen specimens of each species. MSS-PCR lead to a species-specific amplification of a 1,540 bp fragment in L. polyactis, 1,013 bp in A. nibe, 474 bp in L. crocea, and 182 bp in P. elongates, respectively. The four different sizes of each PCR product can be quickly and easily detected by single gel electrophoresis. The sensitivity of the MSS-PCR of the DNA was up to $0.1ng/{\mu}l$ as a starting concentration for the four different species tested. These results suggest that MSS-PCR, with species-specific primers based on SNP, can be a powerful tool in the rapid identification of the four drums species, L. polyactis, L. crocea, A. nibe, and P. elongates.
Keywords
COI; drums; molecular identification; multiplex PCR; species specific primer;
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