To identify the genuine species of Hirudo (

Whitmania pigra

Hirudo nipponica

)

DNA molecular method was established. Based on mitochondrial cytochrome

c

oxidase subunit Ⅰ(COⅠ) gene

speciesspecific primers were designed for

W. pigra

and

H. nipponica

respectively. Using three species-specific primers

PCR amplification was performed for the genuine species and the common adulterant

Poecilobdella manillensis

Lesson

as well as the Naoxintong capsules. Results showed that both primers WF1R2 and WF2R2 could specifically amplify the DNA of

W. pigra

with amplicons of around 200 bp

while the primers HF1R2 could specifically amplify the DNA of

H. nipponica

to generate amplicons of 142 bp. Besides

no amplification was obtained in the adulterant

P. manillensis

with above three pairs of primers. In addition

amplicons around 200 bp were obtained in Naoxintong capsules using primers WF1R2 or WF2R2

with the same sequences as

W. pigra

which is the genuine species that the company used. These results indicated that without sequencing

the primers WF1R2 (or WF2R2) and HF1R2 could specifically amplify

W. pigra

and

H. nipponica

respectively. Furthermore

the primers WF1R2 and WF2R2 could be applied to identify the genuine species in Naoxintong capsule. This DNA molecular identification method is simple

accurate

with high specificity and sensitivity

thus it could be used as a supplement to the conventional method for origin identification and further improve the quality control of Hirudo and the Hirudo in Naoxintong capsule