The development and application of mini-barcodes from mitochondrial DNA for identifying medicinal leeches from traditional medicines.

Species-specific efficacy necessitates accurate identification of medicinal leeches, but standard DNA barcoding often fails with degraded DNA from traditional medicines. This deficiency highlights the need for mini-barcoding. This study aimed to develop and validate mini-barcode markers for three Chinese Pharmacopoeia-listed leech species: Whitmania pigra, Whitmania acranulata and Hirudo nipponia. Four novel mini-barcode primer sets (ND1F1/R1, 12SF1/R1, 16SF1/R1 and COX1F1/R1) were developed and validated using seven morphologically identified specimens and subsequently tested on 16 commercial leech products. DNA extractions were performed using both single-tube and column purification kits, with the latter yielding superior DNA quality and meeting the requirements for following PCR amplification. The PCR results confirmed the validation of four candidate mini-barcodes targeting specific genetic regions, which produced results in 13 out of 16 commercial leech products. Mini-barcode sequences from morphologically identified W. pigra specimens exhibit > 95% identity to the complete ND1, 12S rDNA, 16S rDNA, and COX1 sequences (EU304459), whereas sequences from H. nipponia and W. acranulata show < 85% identity, and among leech-derived products only the proprietary Chinese medicine Maxuekang exhibits lower identity. Both the optimal partition of ASAP and phylogenetic tree identified three distinct groups correlating with the morphological species: W. pigra, W. acranulata, and H. nipponia. Mislabeled species have been uncovered in proprietary Chinese medicine, notably the claimed Hirudo nipponia, which was replaced by W. pigra. The results highlight the value of mini-barcodes in enhancing product quality control and offer a reliable method for accurate species identification in traditional and commercial leech-based medicines. This advance supports safer and more effective utilization of medicinal leeches and advocates for their integration into regulatory standards.