Development of a mitochondrial mini-barcode and its application in metabarcoding for identification of leech in traditional Chinese medicine.

In Traditional Chinese Medicine (TCM), the medicinal leech is vital for treatments to promote blood circulation and eliminate blood stasis. However, the prevalence of counterfeit leech products in the market undermines the quality and efficacy of these remedies. Traditional DNA barcoding techniques, such as the COI barcode, have been limited in their application due to amplification challenges. This study identified high variability in the 16 S rRNA gene within the mitochondrial genome across five leech species, leading to the development of a novel 219 bp mini-barcode. Compared with the traditional COI barcode, our mini-barcode showed remarkable identification efficiency, classifying 142 out of 147 leech samples from fresh and processed materials. In contrast, the COI barcode could only successfully identify 79 out of the 147 samples. In the case of seven batches of leech decoction pieces, the mini-barcode identified six, whereas the COI barcode only recognized one. Additionally, the mini-barcode effectively discerned five leech species within Chinese patent medicines when combined with metabarcoding technology. These results confirm the mini-barcode's potential as a reliable tool for rapidly and precisely identifying leech species in TCM products.