基于环介导等温扩增技术的中药材蕲蛇真伪鉴别方法的建立

Authentication of Agkistrodon acutus based on loop-mediate isothermal amplification

目的:建立基于环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)的蕲蛇快速检测方法,实现中药蕲蛇的可视化快速鉴定。方法:依据《中华人民共和国药典》收录的蕲蛇特异性序列设计4条LAMP引物(2条内引物、2条外引物),建立蕲蛇LAMP的鉴别方法,对比《中华人民共和国药典》收录的聚合酶链式反应(polymerase chain reaction, PCR)方法的检测结果,评价LAMP的鉴别方法的特异性和灵敏度,并采用LAMP鉴别方法和PCR方法鉴别市售蕲蛇的真伪。结果:使用该引物建立的蕲蛇LAMP鉴别方法对蕲蛇对照药材和4份市售蕲蛇样本产生扩增,对1份市售蕲蛇样本和8种蕲蛇混淆品样本无扩增,与PCR鉴别结果一致,说明蕲蛇的LAMP鉴别方法具有良好的特异性。LAMP检测的灵敏度在DNA水平上达到0.004 ng·μL^(-1),PCR检测的灵敏度为0.4 ng·μL^(-1),因而LAMP检测具有较高的灵敏度。结论:本研究建立的LAMP检测方法可以应用于中药材蕲蛇真伪的快速鉴别。

Objective:To establish a rapid visual identification method for Agkistrodon acutus based on loop-mediated isothermal amplification(LAMP).Methods:The LAMP assay for the detection of Agkistrodon acutus was developed by designing four primers including two inner primers and two outer primers which according to the specific sequence of Agkistrodon acutus contained in the Chinese Pharmacopoeia.Specificity and sensitivity of the LAMP assay was validated by comparing the polymerase chain reaction(PCR)method included in the Chinese Pharmacopoeia,and using the LAMP assay and the PCR to identify the authenticity of agkistrodon acutus on the market.Results:The LAMP assay established by using those primers showed good performance on specificity and sensitivity.Amplification could be observed in reference crude drug of Agkistrodon acutus and four samples of Agkistrodon acutus from the market,and there was no amplification in one sample of Agkistrodon acutus from the market and eight common adulterants,which was consistent with the identification results of PCR.The sensitivity of the LAMP assay was 0.004 ng·μL^(-1)and the sensitivity of the PCR assay was 0.4 ng·μL^(-1),so that the LAMP assay showed higher sensitivity.Conclusion:The result demonstrated that the specific LAMP assay can be used for rapid detection of Agkistrodon acutus.