Identification of traditional Chinese medicinal pipefish and exclusion of common adulterants by multiplex PCR based on 12S sequences of specific alleles.

We aimed to establish a rapid and accurate allele-specific diagnostic Polymerase Chain Reaction (PCR) method for medicinal pipefish. To achieve this, pipefish genomic DNA was extracted, sequenced bi-directionally, and the data were analyzed. On this basis, specific identification primers were designed and a facile multiplex PCR system was established and optimized. Phylogenetic tree analysis showed that the six species of pipefish were strictly clustered in separate single branches. The reaction was optimized for ease of application, to be used in a reaction volume of 20 μL with template DNA amounts in the range of 5-100 ng, and an annealing temperature from 43 to 55 °C. The reactions conducted using authentic samples of Syngnathoides biaculeatus, Solenognathus hardwickii, and Syngnathus acus produced clear single DNA bands of 240 bp, 318 bp, and 139 bp, respectively. The observed amplicons correspond very well to the specific identification primers HLN, HLD, HLJ, as designed. Thus, it can be concluded that our identification system is specific and stable, and can be used to quickly and accurately identify complex multi-source pipefish samples. We hope that the system will not only ensure the quality of traditional Chinese medicinal ingredients, but also help conservation efforts by offering a quick and easy identification method for pipefish.