鼠疫耶尔森氏菌环介导等温扩增检测方法的建立

Development of loop-mediated isothermal amplification method for detection of Yersinia pestis

目的:将环介导等温扩增技术(LAMP)应用于鼠疫耶尔森氏菌的快速检测。方法:针对鼠疫耶尔森氏菌F1基因设计特异性引物,进行LAMP扩增。应用LAMP技术对鼠疫菌和非鼠疫菌进行检测以确定其特异性;对倍比稀释鼠疫菌液进行检测以确定其灵敏度,并与常规PCR方法进行比较。结果:对10种非鼠疫菌进行LAMP扩增,均为阴性结果,证明该方法具有很高的特异性。LAMP方法最低检出量为20个鼠疫菌,比常规PCR方法的灵敏度高10倍。通过加入染料进行LAMP扩增可直接观察结果。结论:本方法检测鼠疫耶尔森氏菌特异性强、灵敏度高,并且操作简便、结果易于判定,有望发展成为快速检测鼠疫耶尔森氏菌的有效手段。

Objective：To develop a method of 1oop-mediated isothermal amplification（LAMP） for detection of Yersinia pestis.Methods： According to conserved F1 gene of Yersinia pestis and principle of LAMP,a set of LAMP reaction system containing four primers were designed.Strains of Yersinia pestis and non-Yersinia pestis were detected with LAMP to evaluate the specificity of the method.The dilution of the Yersinia pestis was detected with LAMP and PCR to evaluate the sensitivity of the method.Results： 10 non-Yersinia pestis strains were found all negative by LAMP,performing high specificity of this method.The detection limit of this LAMP assay was about 20 yersinia pestis strains per reaction.The sensitivity of this LAMP assay was 10-fold higher than regular PCR.A visual detection approach with addition of dye was feasible to interpret the positive or negative results.Conclusion： The genetic diagnostic method of Yersinia pestis based on LAMP is rapid,sensitive and specific.This method may be the potential effective way for detection of Yersinia pestis on the scene.