담수산 복족류 (Gastropoda: Architaenioglossa) 3종 간 신속한 Triplex PCR 종 판별 방법

Abstract

This study designed species-specific primers for quick and convenient molecular biological species identification of three species belonging to Architaenioglossa widely distributed in Korea and developed a single triplex PCR primer set by verifying specificity and reproducibility. Species-specific primers were designed to amplify species-specific bands (Pomacea canaliculata, 223 bp; Sinotaia quadrata, 261 bp; Cipangopaludina chinensis malleata, 431 bp) in consideration of positions representing genetic variation within species and between species on the cytochrome c oxidase subunit 1 (co1) gene base sequence of mitochondrial DNA. In addition, conventional PCR verification experiments were performed according to the amplification cycle and concentration range of genomic DNA (gDNA) to verify the amplification efficiency when configuring the triplex PCR primer set. As a result, considering the PCR reaction cycle and sample amount, the minimum PCR reaction cycle and gDNA concentration were found to be each 25 cycles and 10 ng/μL. Therefore, it is judged that the triplex PCR primer set between the three species belonging to Architaenioglossa developed in this study can contribute to accurate species identification in the industrial field with a quick and simple analysis method at the conventional PCR level.