聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）鉴定川贝母药材的方法优化

目的：改进中国药典2015年版中川贝母聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）的鉴定方法，以防止真菌污染对结果的影响。方法：采用中国药典2015年版中PCR-RFLP方法对川贝母样品进行检测，对PCR产物电泳中出现的杂带进行了序列分析，确认来源于真菌的扩增产物。重新设计PCR-RFLP中的扩增引物，将下游引物设计在贝母与真菌序列差异较大的ITS2区域。对改进后的PCRRFLP方法进行了验证。结果：用含新引物的PCR-RFLP鉴定时PCR产物未出现杂带，改进方法鉴定的准确性优于中国药典2015年版中给出的方法。结论：改进的PCR-RFLP方法可以对川贝母进行鉴定，同时防止了真菌污染产生的杂带对结果判断的影响。

Objective: To improve PCR-RFLP identification method of Fritillariae Cirrhosae Bulbus in Chinese Pharmacopoeia(2015 edition)for preventing the interference of fungus contamination. Methods: Fritillariae Cirrhosae Bulbus samples were detected using PCR-RFLP method in Chinese Pharmacopoeia. And the sequences of unexpected DNA bands in electrophoresis after PCR were analyzed and confirmed to be amplification products of fungus. Then the primers for PCR-RFLP were redesigned by putting the downstream primer in ITS2 area where Fritillaria L. and fungus had low homology. The improved method was validated. Results: There was only one specific band in electrophoresis after PCR and the improved PCR-RFLP gave more accurate result than the one in Chinese Pharmacopoeia. Conclusion: The improved PCR-RFLP method could be used for identification of Fritillariae Cirrhosae Bulbus and prevent the interference of unspecific DNA band brought by fungus contamination.