Specific authentication of Hippocampus based on SNP markers

Seahorses, which are used as traditional Chinese medicine, have large market demands and premium price. Because of overfishing, many seahorse species are endangered and listed in IUCN Red List. The purpose of this study was to develop a simple, rapid and reliable molecular method for specific authentication of seahorse species. Cytochrome c oxidase subunit I (COI) sequences were analyzed for molecular authentication of seahorses in this study. Many single nucleotide polymorphism (SNP) sites were detected among COI sequences of nine seahorse species. Based on the SNP sites, four pairs of modified specific primers were designed respectively for authentication of Hippocampus kuda, Hippocampus japonicus, Hippocampus trimaculatus and Hippocampus kelloggi using allele-specific PCR. Moreover, a multiplex allele-specific PCR for simultaneous authentication of H. kuda, H. japonicus and H. trimaculatus was also developed. The study showed that allele-specific PCR required no sequencing and electrophoresis, and the only necessity is to add fluorochrome into PCR products. Hence the whole authentication procedure only takes about 1.5 h. This work provides an effective method for rapid and reliable identification of seahorse species.