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目的建立并应用RT-LAMP检测新甲型H1N1、季节性H1N1和季节性H3N2亚型流感病毒及乙型流感病毒。方法通过在线软件Primer Explorer V5设计针对新甲型H1N1、季节性H1N1和季节性H3N2亚型及乙型流感病毒基因的保守区引物,建立RT-LAMP检测方法,对2012年-2013年采集的疑似咽拭子标本采用real-time PCR和RTLAMP法检测。结果 615份标本中,real-time PCR法检出295份阳性,RT-LAMP法检出294份阳性,2种方法的检测结果差异无统计学意义(P>0.05)。2种方法所建立的季节性H1N1、季节性H3N2、新甲型H1N1、乙型的检测体系只与相应亚型流感病毒呈阳性反应,而对禽流感病毒H5、禽流感病毒H9、RSV、RV、EV71扩增结果为阴性。RT-LAMP检测方法具有良好的敏感性和特异性。结论 RT-LAMP法使用水浴锅进行等温扩增,结果在可见光或紫外灯下直接肉眼观察。RT-LAMP检测方法具有高度的敏感性、准确性、高特异性,而且简单、快速,比real-time PCR更适合应用于基层疾病预防控制中心和医院。
Objective To establish and use RT-LAMP to detect influenza A (H1N1), seasonal H1N1 and seasonal H3N2 influenza viruses and influenza B viruses. Methods Primer Explorer V5 was used to design conservative region primers for the novel H1N1, seasonal H1N1 and seasonal H3N2 subtypes and influenza B virus genes. RT-LAMP detection was established to detect the suspected Throat swabs were detected by real-time PCR and RTLAMP. Results Among the 615 samples, 295 were positive by real-time PCR and 294 by RT-LAMP. There was no significant difference between the two methods (P> 0.05). The detection methods of seasonal H1N1, seasonal H3N2, new H1N1 and B of the two methods were only positive with the corresponding subtype of influenza virus, but not of H5, H9, RSV, RV , EV71 amplification was negative. RT-LAMP detection method has good sensitivity and specificity. Conclusion The RT-LAMP method was used for isothermal amplification in a water bath, and the result was visually observed under visible light or UV light. The RT-LAMP assay is highly sensitive, accurate, and highly specific, and is simple, rapid and more suitable for use in primary disease prevention and control centers and hospitals than real-time PCR.