环介导核酸恒温扩增结合试纸条技术快速筛查沙门菌的方法

Rapid screening of Salmonella by loop-mediated isothermal amplification combined with lateral flow dipstick

[目的]探讨环介导核酸恒温扩增(LAMP)结合试纸条技术(LFD)快速筛查沙门菌的可行性。[方法]从GenBank上下载沙门菌invA基因序列,用DNAMAN软件同源性比对后,在其保守区设计LAMP扩增引物,建立LAMP-LFD恒温扩增快速检测技术,优化反应条件,用荧光聚合酶链反应(PCR)法平行检测加以验证。[结果]本方法对沙门菌invA基因检测具高度特异性,对构建的阳性质粒DNA灵敏度达到1.0×10^(1)拷贝/μL,其临床标本(肛拭子)检出率与荧光PCR法相同。实验操作简便、快速(30 min)。[结论]本研究建立的LAMP-LFD有望成为沙门菌快速筛查方法之一。

[Objective]To establish a specific and sensitive method using loop-mediated isothermal amplification for rapid screening of Salmonella.[Methods]The invA gene sequence of Salmonella was downloaded from GenBank.After homology comparison with DNAMAN software,amplification primers were designed in the conserved region,and a LAMP-LFD detection method was established.The reaction system was optimized,and the specificity and sensitivity of the method were verified.[Results]The sensitivity of this method to detect Salmonella DNA was up to 1.0×10^(1) copies/μL.The positive rate of anal swabs was the same as that of fluorescent PCR.Meanwhile,LAMPLFD was easy to operate and did not need expensive instruments.The detection result could be obtained within 30 minutes.[Conclusion]The LAMP-LFD method established in this study is rapid,simple,sensitive and specific,which is suitable for rapid screening of Salmonella.