Development of Rapid Identification Method for Four Species of Ark Shells (Bivalvia: Arcoidae) Bivalves Using Multiplex PCR
Yong Hwi Kim, Ho-Seop Han, Bong Han Yun, Jong Yeon Park In Gug Baek, In-Chul Bang
Department of Biology, College of Natural Sciences, Soonchunhyang University, Asan, Chungnam 3538, Republic of Korea Aqua Biotech Co., Ltd., 62-6 -24, Yuseong-daero 84beon-gil, Yuseong-gu, Daejeon, 3409, Republic of Korea
A multiplex PCR primers (the species-specific primers) set using molecular biological methods targeting four species of ark shells (Anadara kagoshimensis, Tegillarca granosa, Anadara broughtonii and Cucullaea labiata) distributed in Korea were built, and a rapid and accurate species identification method was developed. The species-specific primer was designed within the nucleotide sequence of about 573 bp corresponding to the cytochrome c oxidase subunit 1 (co1) gene region of mitochondrial DNA, and considering the single nucleotide polymorphism (SNPs) representing inter-species variation excluding intra-species variation, it was designed with an interval of 80-100 bp for each species. As a result, it was confirmed that species-specific bands were formed in the order of A. kagoshimensis (160 bp), T. granosa (245 bp), A. broughtonii (351 bp), and C. labiata (471 bp). In addition, in order to measure the limit of multiplex PCR amplification according to the concentration of genomic DNA (gDNA) and the number of PCR amplification repetitions, 15 experimental groups were divided and measured. As a result, when the number of amplification repeats was 25 cycles, the concentration of gDNA was detected down to a minimum of 1 ng/μl. Therefore, the multiplex PCR primer set for four species of ark shells developed in this study is expected to be of great help in accurately and rapidly identifying species in the field, it is thought that economic damage and academic errors caused by misidentification can be prevented.
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