逆转录环介导等温扩增技术检测H7N9禽流感病毒基因

Rapid Visual Detection of H7N9 avian influenza virus by use of reverse transcription loop-mediated isothermal amplification

目的建立检测H7N9禽流感病毒基因的逆转录环介导等温扩增(reverse transcription Loop-mediated isothermal amplification,RT-LAMP)方法,并初步评估应用。方法设计、合成H7N9禽流感病毒血凝素(HA)基因、神经氨酸酶(NA)基因的引物组,通过优化参数,建立RT-LAMP反应体系,使用梯度稀释的标准品测试该体系检测灵敏度,并使用对比毒株测试特异性,再对142份临床标本进行实样检测。结果成功建立了检测H7N9禽流感病毒HA基因、NA基因的RT-LAMP方法,可直接肉眼观察判读结果,检测其HA基因、NA基因的敏感性分别达到10拷贝每反应、5拷贝每反应,对其他常见呼吸道病原无交叉反应;在测序证实阳性的临床样本中,对于病毒HA基因、NA基因检出率分别为100%、92.68%。结论逆转录环介导等温扩增技术检测H7N9禽流感病毒基因,具有简单、快速、灵敏度高,检测结果可视化,无需特殊设备等优点,在禽流感病原监测上有一定应用前景。

Objective To develop a one-step RT-LAMP method for rapid detection of HA gene and NA gene of H7N9 virus.Methods Two sets of specific primerswere designed and synthesized according to HA and NA genes of H7N9 virus. The reaction parameters were optimized to improve the RT-LAMP assay for the rapid detection of H7N9 virus.This study also tested the sensitivity and specificity of the RT-LAMP assay,the minimum detection limit of which was evaluated using the gradient dilution RNA templates in vitro transcription. Moreover,the RT-LAMP was applied to 142 clinical specimens for testing. Results The RT-LAMP method for detecting the HA gene and NA gene of H7N9 avian influenza virus was successfully established.The results showed that the detection limit for H7 gene is around 10 copies per reaction,which is similar to that of the real time PCR,whereas counterpart of N9 gene is 5 copies perreaction in 100-folds higher sensitivity than the realtime PCR method. The RT-LAMP assay also had good specificity.In the positive specimens confirmed by sequencing,the RT-LAMP detection rate of viral HA gene and NA gene were 100% and 92. 68%,respectively. Conclusion Considering RT-LAMP’s simplicity in operation and high sensitivity,there is potential use in clinical diagnosis and surveillance of H7N9 virus.