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KMID : 0613820170270070746
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Journal of Life Science
2017 Volume.27 No. 7 p.746 ~ p.753
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Development of a Multiplex PCR Assay for Rapid Identification of Larimichthys polyactis, L. crocea, Atrobucca nibe, and Pseudotolithus elongates
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Noh Eun-Soo
Lee Mi-Nan
Kim Eun-Mi
Park Jung-Youn
Noh Jae-Koo
An Cheul-Min
Kang Jung-Ha
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Abstract
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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℃. 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 0.01 ng/μl 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.
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KEYWORD
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COI, drums, molecular identification, multiplex PCR, species specific primer
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