宽体金线蛭的特异性PCR分子鉴定

Specific PCR molecular identification of Whitmania pigra

关键词:  宽体金线蛭  特异性聚合酶链式反应  分子鉴定  荧光检测

Key words:  Whitmania Pigra  specific PCR  molecular identification  fluorescence detection

摘要:

[目的] 实验旨在通过寻找宽体金线蛭单核苷酸多态性（SNP）位点，设计宽体金线蛭的特异性鉴别引物，建立宽体金线蛭特异性聚合酶链式反应（PCR）检测方法，实现对宽体金线蛭及其常见混伪品的快速鉴别。[方法] 通过比对宽体金线蛭与其常见混伪品的细胞色素C氧化酶亚基I（COI）序列，寻找宽体金线蛭COI基因序列内SNP位点，设计特异性鉴别引物。[结果] 进行PCR后仅宽体金线蛭能够扩增得到149 bp的条带，混伪品不能扩增出明显目的条带。向扩增产物中加入SYBR Green I核酸染料染色，宽体金线蛭样品在紫外灯下显黄绿色荧光，混伪品不显荧光。[结论] 该方法能够准确快速鉴别宽体金线蛭与其常见混伪品，为中药材市场上宽体金线蛭的快速鉴别提供技术支持。

Abstract:

[Objective] To design the specific identification primers of Whitmania Pigra by searching for single nucleotide polymorphism (SNP) sites,establish specific polymerase chain reaction (PCR) detection technology,and realize rapid identification of Whitmania Pigra and its common adulterants.[Methods] By comparing the cytochrome c oxidase I (COI) sequences of Whitmania Pigra and their common adulterants,searchinging for SNP sites in COI gene sequence of Whitmania Pigra,and then designing specific identification primers.[Results] After the PCR process,the Whitmania Pigra could amplify a 149 bp band,whereas the adulterants can't get the obvious target band. When the amplified product was stained with SYBR Green I dye,the Whitmania Pigra sample showed yellow-green fluorescence under UV light,whereas the adulterants did not show fluorescence.[Conclusion] This method can identify Whitmania Pigra and its common adulterants accurately and quickly,which can provide technical support for the rapid identification of Whitmania Pigra in Chinese medicinal materials markets.