Advancing Species Identification in Traditional Chinese Medicine: A Novel Mini-Barcode from Mitochondrial Genomes Combined with Meta-barcoding Technology for Leech Authentication

In Traditional Chinese Medicine (TCM), the medicinal leech is a vital component for treatments aimed at promoting blood circulation and eliminating blood stasis. However, the prevalence of counterfeit leech products in the market undermines the quality and efficacy of these remedies. Conventional DNA barcoding techniques, such as the COI barcode, have proven inadequate for TCM species identification due to amplification challenges. This study introduces a novel 219bp mini-barcode, derived from the mitochondrial genome's 16S rRNA gene, exhibiting high variability among five leech species. We also used primers 741F-943R to enhance the identification process. Compared with the traditional COI barcode, our mini-barcode showed remarkable identification efficiency, accurately classifying 144 out of 147 leech samples from fresh and processed materials. In the case of seven batches of leech decoction pieces, the mini-barcode identified six, whereas the COI barcode only recognized one. Additionally, when combined with meta-barcoding technology, the mini-barcode effectively discerned five leech species within Chinese patent medicines. These results confirm the mini-barcode's potential as a reliable tool for the rapid and precise identification of leech species in TCM products.