首页|宽体金线蛭COI基因特异性引物的设计及验证

宽体金线蛭COI基因特异性引物的设计及验证

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目的 建立一种宽体金线蛭的聚合酶链式反应鉴别方法,实现对宽体金线蛭及其常见混伪品的快速鉴别.方法 以细胞色素c氧化酶亚基Ⅰ序列为基础,通过分析宽体金线蛭单核苷酸多态性位点,设计宽体金线蛭的特异性鉴别引物.对引物稳定性、引物特异性及灵敏度进行考察确认,同时将其应用于含水蛭的中成药中进行验证.结果 使用宽体金线蛭特异性引物KT1-3或KT1-4均可特异性扩增宽体金线蛭的DNA,产物大小分别为235 bp和266 bp,而在其他非宽体金线蛭样品中无扩增条带.该方法对宽体金线蛭DNA的最低检测限为0.01 ng.使用该特异性引物对可在脑血栓片模拟制剂和部分市售脑血栓片中检测到宽体金线蛭.结论 建立的DNA分子鉴定方法中宽体金线蛭引物对特异性强、灵敏度高,可作为水蛭药材传统基原鉴定方法的补充,为宽体金线蛭的快速鉴别提供支持.
Design and Validation of Specific Primers for COI Gene of Whitmania pigra Whitman
Objective To establish a polymerase chain reaction(PCR)method for rapid identification of Whitmania pigra Whitman and its common counterfeits.Methods A set of specific primers were designed for the identification of Whitmania pigra Whitman,based on the sequence of cytochrome c oxidase subunit Ⅰ(COI)and single nucleotide polymorphism(SNP)mutation sites.The stability,specificity and sensitivity of the primers were confirmed,and the application of the primers in Chinese patent medicine containing leech was verified.Results The specific primers named KT 1-3 or KT 1-4 were used to amplify the deoxyribonucleic acid(DNA)of leech,and the products were 235 bp and 266 bp,respectively.The LOD of this method was 0.0 1ng.The specific primer pair can be used to detect Whitmania pigra Whitman in simulated preparations of Naoxueshuan tablet and some commercial Naoxueshuan tablets.Conclusion The primers of the DNA molecular identification method has high specificity and sensitivity,which can be used as a supplement to the traditional identification method of leech medicinal materials and provide support for the rapid identification of Whitmaniapigra Whitman.

DNA barcodeWhitmania pigra Whitmanspecies-specific primersadulterationorigin identification

潘爽、刘金花、孙巧巧、李鹏举

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济宁市食品药品检验检测研究院生物测定科,山东济宁 272073

DNA条形码 宽体金线蛭 特异性引物 掺伪 基原鉴定

2022年山东省药品质量风险监测项目

局函[2022]174号

2024

现代中药研究与实践
安徽中医药高等专科学校,

现代中药研究与实践

CSTPCD
影响因子:0.409
ISSN:1673-6427
年,卷(期):2024.38(2)