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.

Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.

Develops short mitochondrial DNA mini-barcodes for identifying medicinal leech species in highly processed traditional medicines, with PCR-validated species discrimination.

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