Specific DNA identification of Pheretima in the Naoxintong capsule

BACKGROUND: Pheretima is a minister drug in Naoxintong capsule (NXTC), a well-known traditional Chinese medicine (TCM) formula for the treatment of cardiovascular and cerebrovascular diseases. Owing to the loss of morphological and microscopic characteristics and the lack of recognized chemical marker, it is difficult to identify Pheretima in NXTC. This study aims to evaluate the feasibility of using DNA techniques to authenticate Pheretima, especially when it is processed into NXTC. METHODS: DNA was extracted from crude drugs of the genuine and adulterant species, as well as nine batches of NXTCs. Based on mitochondrial cytochrome c oxidase subunit I (COI) gene, specific primers were designed for two genera of genuine species, Metaphire and Amynthas, respectively. PCR amplification was performed with the designed primers on crude drugs of Pheretima and NXTCs. The purified PCR products were sequenced and the obtained sequences were identified to species level with top hit of similarity with BLAST against GenBank nucleotide database. RESULTS: Primers MF2R2 and AF3R1 could amplify specific DNA fragments with sizes around 230–250 bp, both in crude drugs and NXTC. With sequencing and the BLAST search, identities of the tested samples were found. CONCLUSION: This study indicated that the molecular approach is effective for identifying Pheretima in NXTC. Therefore, DNA identification may contribute to the quality control and assurance of NXTC.