甲_3型流行性感冒病毒环介导逆转录等温扩增检测方法的建立与应用

Development and Application of Loop-mediated Isothermal Amplification Assay(LAMP)for Detection of Influenza Virus Subtype H_3

目的建立适用甲3型流行性感冒(流感)病毒核酸检测的环介导逆转录等温扩增(RT-LAMP)方法,为基层实验室流感快速诊断服务。方法对甲3型流感病毒设计两对环介导特异性引物与一个环引物,采用环介导RT-LAMP技术对甲3型流感病毒核酸进行等温扩增,并对反应体系进行优化。结果采用RT-LAMP技术检测甲3型流感病毒核酸,敏感度可达0.01TCID50/反应,与荧光定量逆转录-聚合酶链反应(RT-PCR)的敏感度相一致,且对H1和H5亚型流感病毒、乙型流感病毒、呼吸道合胞病毒、麻疹病毒、风疹病毒、流行性腮腺炎病毒、副流感病毒与腺病毒无交叉反应,具有良好的特异性。采用该方法检测58份临床样本,病毒分离的阳性率为37.9%,而荧光定量RT-PCR与RT-LAMP的阳性率分别为53.45%和51.72%,敏感度明显高于通常的细胞病毒分离。结论甲3型流感病毒环介导RT-LAMP检测方法,不仅快速特异、敏感性强,而且简便价廉,适于基层实验室使用。

Objective To provide service of rapid diagnosis of influenza virus in the lab at grass root level by setting up the Loop-mediated isothermal amplication assey to detect the nucleic acids of infmenza virus sbutype H3. Methods Two pairs of specific primers and a loop primer were designed. Nucleic acids of influenza virus H3 were amplified using reverse-transcription and loopmediated isothermal amplification technology（RT-LAMP）. The reaction systems were optimized. Results Sensitivity of the RTAMP assay was 0.01TCID50/reaction which was as same as that of the real-time RT-PCR assay. There was no crossing-reaction with other aspiratory viruses including influenza virus subtype H1 and H5,influenza B,Respiratory Syncytial Virus（RSV）,Measles virus,Rubella virus, Mumps virus,adenovirus and para-influenza virus which indicated the good specificity of the RT-LAMP. 58 of clinical samples were analyzed by real-time PCR and RTLAMP. The positive rate of virus isolation was 37.9%. The real-time PCR and RT-LAMP assays were 53.45% and 51.72% of respectively. The sensitivity of two assey was 100 times higher than that of the viral isolation. Conclusions Thit loop-mediated isothermal amplification assay for detection of influenza virus H3 was not only rapid, specific and sensitive, but also simple and cheap. It was very suitable for the labs at grass root level.