微芯片电泳结合多重PCR法在中药材水蛭品种特异性鉴定中的应用

目的：通过特异性引物的聚合酶链式反应（PCR）法结合微芯片电泳检测的方法，特异性鉴别宽体金线蛭、日本医蛭、菲牛蛭3种水蛭品种。方法：针对宽体金线蛭、日本医蛭、菲牛蛭3个品种的线粒体同源序列，设计区分正伪品水蛭以及分别鉴定3个品种水蛭样品的特异性引物；通过PCR和微芯片电泳检测的方法验证所设计引物的特异性；将验证得到的特异性引物进行整合，建立3个品种的特异性引物用于三重PCR的方法。结果：确定了用于区分宽体金线蛭、日本医蛭、菲牛蛭3个品种中正伪品的特异性引物KA；确定了宽体金线蛭特异性引物KT，日本医蛭特异性引物RI，菲牛蛭特异性引物FN；建立了3种特异性引物进行三重PCR鉴别3个品种水蛭药材的方法。结论：通过特异性引物结合三重PCR的方法，可以同步鉴定宽体金线蛭、日本医蛭、菲牛蛭的人为预混样品，鉴定结果准确，且用于三重PCR的特异性引物不存在引物之间相互结合或非特异性扩增的现象。

Objective: Using the method of polymerase chain reaction (PCR) by particular primer combined with microchip electrophoresis detection,to specifically identify 3 species of Hirudo,Whitmania pigra,Hirudo nipponia and Poecilobdella manillensis. Methods: Particular primers were designed to distinguish the genuine with fake and specifically identify Whitmania pigra,Hirudo nipponia and Poecilobdella manillensis based on the mitochondrial homologous sequence. Specificity of the particular primers was verified by the method of PCR combined with microchip electrophoresis. The three verified particular primers were integrated and a triple PCR method was set up for the 3 Hirudo. Results: KA was verified as the particular primer for identification of the genuine and fake. KT was verified as the particular primer for the identification of Whitmania pigra,RI was verified as the particular primer for the identification of Hirudo nipponia,and FN was verified as the particular primer for the identification of Poecilobdella manillensis. Finally,a triple PCR method to identify the three species of Hirudo was established. Conclusion: Through the method of particular primers combined with PCR,the artificial mixed sample of Whitmania pigra,Hirudo nipponia and Poecilobdella manillensis could be identified synchronously. The identification results were accurate,and combining of particular primers or nonspecific amplification was not found.

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