环介导等温扩增技术检测鼠疫耶尔森菌的敏感性和特异性研究

Sensitivity and specificity of loop-mediated isothermal amplification in detection of Yersinia pestis

为观察环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术能否适用于我国不同疫源地鼠疫耶尔森菌所有基因组型的检测,本研究建立了一种基于3a靶序列设计特异性引物快速检测鼠疫耶尔森菌的LAMP方法。选择分离自我国11个鼠疫自然疫源地的65株野生代表性鼠疫耶尔森菌株,同时以与其近源的假结核耶尔森菌、小肠结肠炎耶尔森菌及非近源的大肠埃希菌为对照菌株,观察其敏感性和特异性。结果显示,该LAMP方法与其他3种鼠疫耶尔森菌常规检测方法的结果一致。65株鼠疫耶尔森菌检测均为阳性,最低检出浓度为20个菌/μL;假结核耶尔森菌、小肠结肠炎耶尔森菌及大肠埃希菌标准菌株反应均为阴性。结果提示,本研究建立的LAMP技术对检测我国各疫源地鼠疫耶尔森菌具有高度特异性,且快速简便,无需特殊仪器,可肉眼判读结果,适用于野外现场鼠疫监测中的快速检测,在偏远地区疫情防控中具有实用价值。

The present paper aims to study the sensitivity and specificity of loop-mediated isothermal amplification(LAMP)in detection of Yersinia pestis(Y.pestis)from natural plague foci in China.The LAMP was established based on the specific primers targeting the 3 agene of Y.pestis.Sixty-five Y.pestis strains isolated from 11 natural plague foci in China were subjected to the study,and Y.pseudotuberculosis,Y.enterocolitica and Escherichia coli(E.coli)were selected as control strains.The results of LAMP detection were consistent with three standard diagnostic methods for the plague-reverse indirect haemagglutination test,colloidal gold immunochromatography assay and conventional polymerase chain reaction method.The 65 Y.pestis strains were all positive,while Y.pseudotuberculosis,Y.enterocolitica and E.coli were negative,suggesting that the established LAMP method was highly specific for Y.pestis.With the advantages of fast,simple and low requirements on equipment,the method has potential in field monitoring and detection in remote areas for plague prevention and control.