Liu Yingkui, Xie Youqun, Wang Yu, Zong YanYan, Liu Yong, Li Chong, Dong Jiajia
School of Life Sciences, Xuzhou Medical University, Xuzhou, 221004, China.
Jiangsu Key Laboratory of Brain Disease Bioinformation, Research Center for Biochemistry & Molecular Biology, Xuzhou Medical University, Xuzhou, 221004, China.
Sci Rep. 2025 Jul 1;15(1):20444. doi: 10.1038/s41598-025-04610-3.
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.
物种特异性功效需要准确鉴定药用蚂蟥，但标准DNA条形码技术在处理传统药物中降解的DNA时常常失效。这一缺陷凸显了微型条形码技术的必要性。本研究旨在开发并验证针对《中国药典》收录的三种蚂蟥物种的微型条形码标记：宽体金线蛭、尖细金线蛭和日本医蛭。开发了四组新型微型条形码引物（ND1F1/R1、12SF1/R1、16SF1/R1和COX1F1/R1），并使用七个经形态学鉴定的标本进行验证，随后在16种市售蚂蟥产品上进行测试。DNA提取使用单管和柱式纯化试剂盒，后者产生的DNA质量更高，满足后续PCR扩增的要求。PCR结果证实了针对特定基因区域的四个候选微型条形码的有效性，这四个微型条形码在16种市售蚂蟥产品中的13种上获得了结果。经形态学鉴定的宽体金线蛭标本的微型条形码序列与完整的ND1、12S rDNA、16S rDNA和COX1序列（EU304459）的同一性>95%，而日本医蛭和尖细金线蛭的序列同一性<85%，在蚂蟥衍生产品中，只有 proprietary Chinese medicine Maxuekang的同一性较低。ASAP的最佳划分和系统发育树均确定了与形态学物种相关的三个不同组：宽体金线蛭、尖细金线蛭和日本医蛭。在 proprietary Chinese medicine中发现了错误标记的物种，特别是声称的日本医蛭，实际上被宽体金线蛭所取代。结果突出了微型条形码在加强产品质量控制方面的价值，并为传统和市售蚂蟥类药物的准确物种鉴定提供了一种可靠方法。这一进展有助于更安全、有效地利用药用蚂蟥，并倡导将其纳入监管标准。
