Identification of Pheretima by Multiplex Allele-specific PCR / 中国实验方剂学杂志

ABSTRACT

Objective: In recent years,with the increase in the commodity price of medicinal pheretima,there have emerged increasing adulterates in the medicine market. Besides,the medicinal materials have mostly lost the main identification features, and are difficult to distinguish. Therefore,it is urgent to establish an accurate and stable method for the identification of pheretima. Method: According to the differences of COI gene DNA sequences among Pheretima aspergillum,Pheretima vulgaris,Pheretima guillelmi,Pheretima pectinifera and adulterants,the variation site was found,the specific primers were designed,the reaction conditions were optimized,and the polymerase Chain reaction(PCR) method for identification was explored and verified in terms of tolerance and feasibility in this study. The specific primers were combined to build multiple PCR systems. An effective,accurate,convenient,highly specific and repeatable Multiplex Allele-Specific PCR identification method was established for identifying medicinal pheretima and its common adulterants. Result: Through the established multiplex PCR reaction system, 366,487,487 and 475 bp of fragments were amplified from DNA templates of P. aspergillum,P. vulgaris,P. guillelmi and P.pectinifera respectively. All of the adulterants were negative by the multiplex PCR assay. The PCR amplification of specific alleles method established in this paper can accurately identify pheretima. Conclusion: The Multiplex Allele-Specific PCR identification method established in this paper can accurately identify medicinal pheretima and its adulterants.