多重连接探针扩增技术检测川贝母掺伪的研究

目的：着重探讨采用新技术检测川贝母的真伪。方法：本研究利用多重连接探针扩增（MLPA）技术特异、敏感等技术优势，以川贝母、平贝母、伊贝母、浙贝母和湖北贝母为研究对象，针对川贝母及非川贝母ITS1序列设计了10条特异探针，建立川贝母掺伪检测及相对定量的MLPA扩增方法。结果：针对探针混合物分别以川贝母及4种非川贝母对照药材为模板，从单个样品中只能扩增出单一扩增峰，表明了探针高特异性。敏感性分析结果表明，MLPA扩增最低检测限度可达到掺伪10%，并且目的扩增峰面积比与掺伪比例基本一致。检测市场抽查样品验证了MLPA扩增方法在川贝母掺伪检测中的适用性，表明该方法可有效检出川贝母药材掺伪，并可反映市场药材掺伪比例。结论：本研究基于MLPA建立的川贝母掺伪检测方法，具有特异性强及敏感性高的特点，可为川贝母药材检验监管提供技术支持，为中药材掺伪检测技术的建立提供参考。

Objective: Current detection technologies for Fritillariae Cirrhosae Bulbus analysis is mostly targeted at true or false identification rather than adulteration.In view of the serious adulteration of this medicine,the study of new technology has been a focus of attention.Methods: Multi-parameter assay based on multiplex ligation-dependent probe amplification(MLPA) technology with the advantages of specificity and sensitivity was used.Ten specific probes targeted at Fritillariae Cirrhosae Bulbus,F.Ussuriensis Bulbus,F.Pallidiflorae Bulbus,F.Thunbergii Bulbus and F.Hupehensis Bulbus,were designed respectively and the relative quantitative method for adulteration was established.Results: The mixture of the ten probes only amplified the specific amplification peak from these five Bulbus Fritillariae templates,indicating the probes had good specificity.The result of sensitivity test showed that the adulteration ratio of F.Cirrhosae Bulbus in one MLPA reaction was up to 10%,and the area ratio of the target amplification peaks was in accordance with the adulteration ratio.Then the adulteration test applicability of F.Cirrhosae Bulbus using MLPA method was verified by testing the samples from the market,and the adulteration ratio of the market-collected F.Cirrhosae Bulbus samples was also reflected.Conclusion: The MLPA method described in this article can effectively detect the adulteration in F.Cirrhosae Bulbus,which provids a technical support for inspection and supervision of this important traditional Chinese medicine.