山西中医药大学学报2026，Vol.27Issue(8)：852-858,870,8.DOI:10.19763/j.cnki.2096-7403.2026.05.17
基于COI条形码与特异性qPCR的两种药典水蛭分子鉴别方法研究
Study on molecular identification methods for two leech species in the pharmacopoeia based on COI barcode and specific qPCR
摘要
Abstract
Objective:To establish a molecular identification method based on the mitochondrial cytochrome c oxidase subunit I(COI)gene to accurately distinguish the two authentic leech species recorded in the Pharmacopoeia of the People's Republic of China(Whitmania pigra and Hirudo nipponica)from their common adulterants,providing technical support for the quality control of leech medicinal materials and clinical medication safety.Methods:Dried and fresh leech samples pur-chased from the market were collected.Total DNA was extracted,target fragments were amplified using COI universal primers,and bidirectional sequencing was performed.Species identification was completed by combining NCBI BLAST comparison,MEGA 11.0 sequence alignment,Kimura 2-parameter(K2P)genetic distance analysis,and the construction of a neighbor-joining(NJ)phylogenetic tree.Based on the fixed differential sites in the COI sequences of the two authentic leech species,species-specific primers were designed using Primer Premier 5.0.Their specificity was validated through conventional PCR and SYBR Green real-time quantitative PCR(qPCR)melting curve analysis.Meanwhile,the PCR reaction conditions were optimized,and the detection sensitivity was examined.Results:Among the 8 samples,S2 was identified as Whitmania pigra with 99.85%sequence identity;S7 and S8 were identified as Hirudo nipponica with 99.10%and 99.39%sequence identity,respectively;the remaining samples were all non-pharmacopoeial adulterants.A total of 101 fixed differential sites were de-tected between the two species,with an interspecific genetic distance of 0.170-0.178,significantly higher than the intraspe-cific genetic distance(≤0.017).The two pairs of specific primers designed,KF/KR and YZF/YZR,both showed good speci-ficity in conventional PCR and qPCR.The melting temperatures of the amplification products were stable at(77.0±0.5)℃ and(79.5±0.5)℃,respectively.The optimized PCR reaction conditions were:annealing temperature 59℃,primer volume 1.5 μl,template volume 2.0 μl,and 30 cycles;the detection limit was 0.02 ng/μl.Conclusion:This study established a set of molecular identification methods for leeches based on the COI barcode and specific qPCR.The methods have the advan-tages of strong specificity,high sensitivity,and simple operation.They can be used for the authenticity identification and quality control of leech medicinal materials and provide a reference for the molecular identification of animal-derived tradi-tional Chinese medicinal materials.关键词
宽体金线蛭/日本医蛭/COI条形码/物种特异性/实时荧光定量PCR/熔解曲线/物种鉴定Key words
Whitmania pigra/Hirudo nipponica/COI barcode/species-specific/real-time quantitative PCR/melting curve/species identification分类
医药卫生引用本文复制引用
王孟虎,张萌,于娇,孟祥松,栗进才,孙一帆,牛倩,魏忠侠,张露平..基于COI条形码与特异性qPCR的两种药典水蛭分子鉴别方法研究[J].山西中医药大学学报,2026,27(8):852-858,870,8.基金项目
安徽省教育厅科学研究项目(2025AHGXZK20162) （2025AHGXZK20162）
安徽省中医药传承项目(2025CCCX078) （2025CCCX078）
安徽省高校优秀拔尖人才培育资助项目(YQYB2024085) （YQYB2024085）
亳州市重点研发计划(bzzc2024057) （bzzc2024057）
亳州学院质量工程项目(2024XJXM041,2025XJXM053) （2024XJXM041,2025XJXM053）
