Li Yingxue, Zhou Yi, Ma Yingying, Xu Rong, Jin Xia, Zhang Chiyu
School of Life Sciences, Shanghai University, Shanghai, China.
Pathogen Discovery and Big Data Center, CAS Key Laboratory of Molecular Virology & Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
Bio Protoc. 2019 Nov 5;9(21):e3415. doi: 10.21769/BioProtoc.3415.
Loop-mediated isothermal amplification (LAMP) has been widely used in the detection of pathogens. However, there are usually numerous variants in one viral pathogen and primers employed in LAMP can hardly match all these variants. The mismatches between the primers and the viral genomes, especially those at the 3'-end of the primers, hinder LAMP reactions, leading to failure of the detection. Here, we present a mismatch-tolerant RT-LAMP protocol, which utilizes the 3'-5' exonuclease activity of the Q5 high-fidelity DNA polymerase to remove potential mismatched bases at the 3'-end of the primers during LAMP amplification. Using HIV-1 as a proof-of-principle, we showed that this protocol could represent a promising tool for accurate detection of genetically unstable viruses in laboratory, hospital and field.
环介导等温扩增技术（LAMP）已广泛应用于病原体检测。然而，一种病毒病原体通常存在众多变体，LAMP中使用的引物很难与所有这些变体匹配。引物与病毒基因组之间的错配，尤其是引物3'端的错配，会阻碍LAMP反应，导致检测失败。在此，我们提出了一种耐错配的逆转录LAMP方案，该方案利用Q5高保真DNA聚合酶的3'-5'核酸外切酶活性在LAMP扩增过程中去除引物3'端潜在的错配碱基。以HIV-1作为原理验证，我们表明该方案可能是在实验室、医院和现场准确检测基因不稳定病毒的一种有前景的工具。
