Rapid Differentiation of Seven Species of Anguilla Using PNA Clamping-based Asymmetric PCR with Fluorescence Melting Curve Analysis

Differentiation of the genus Anguilla is important for phylogenetic studies, ecological monitoring, and food forensics. Although various methods have been developed to identify these species, current methods are time-consuming and limit the number of species that can be detected. The aim of this study was to develop a rapid and convenient method to identify the genus Anguilla . To differentiate among seven commercially important species of Anguilla ( A. japonica , A. anguilla , A. rostrata , A. celebensis , A. pacifica , A. marmorata , and A. bicolor pacifica ), fluorescence melting curve analysis (FMCA) with peptide nucleic acid (PNA) probes were used. One primer set was designed to amplify the mitochondrial cytochrome b gene, and seven PNA probes were designed to identify each species from the cyt b gene. The PNA probes were labeled with four different fluorescent reporter dyes (FAM, HEX, Texas Red, Cy5) for multiplex analysis using two tubes. Based on FMCA, each PNA probe showed the highest Tm value when it was 100% complementary to the target sequence. In conclusion, the PNA-based FMCA method is suitable for differentiating among seven species of Anguilla and should be applicable in various fields, such as global marketing and scientific research.