H3N2亚型犬流感病毒RT-LAMP快速检测方法的建立及应用

Establishment and Application of RT-LAMP for Rapid Detection of H3N2 Canine Influenza Virus

为了快速诊断H3N2亚型犬流感病毒(CIV)感染,根据H3N2亚型CIV的M和HA基因序列,设计了3组LAMP引物,优化反应条件,建立了H3N2亚型CIV的RT-LAMP检测方法。结果表明,RT-LAMP方法能在63℃恒温条件下,在45min内完成扩增,通过反应管中预加的荧光染料颜色的变化实现目视化判定。该方法对犬的常见病原无交叉反应,具有较好特异性;灵敏度是常规RT-PCR方法的100倍。该方法操作简单、快速灵敏、特异性强,适合现场应用,对H3N2亚型CIV的检测具有实际的应用价值。

To diagnose H3N2 Canine Influenza Virus(CIV)rapidly,according to the high conservitive sequences of the M and HA genes of H3N2 CIV,three sets of primers were designed.A rapid visable RTLAMP were developped by optimizing the reaction system and conditions.The results showed that the optimzing reaction temperature and time of CIV RT-LAMP were 63℃and 45 minutes,respectively.RT-LAMP amplification products could be visually inspected according to the colour changing of the fluorescent dyes previously added in.Moreover,that no cross-reactivity was found to other common pathogenic microorganisms proved it’s good specificity.The sensitivity of this method was 100 times higher than that of a standard RT-PCR.The above-mentioned results showed that the developed RT-LAMP in this study was more convenient,rapid,highly sensitive and specific than traditional methods.It is more suitable for field testing and has good pactical application value for diagnosing H3N2 CIV.