Objective:

2

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:

2

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:

2

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:

2

The Multiplex Allele-Specific PCR identification method established in this paper can accurately identify medicinal pheretima and its adulterants.