逆转录环介导等温核酸扩增技术(RT-LAMP)在H5N1禽流感病毒基因检测中的应用

Evaluation of Reverse Transcription Loop-mediated Isothermal Amplification for Detection of Avian Influenza A H5N1 Virus

建立一种便捷、灵敏的检测方法,即逆转录环介导等温核酸扩增技术(RT-LAMP)用于H5N1亚型禽流感病毒基因检测。该技术使用特异对应于靶序列中8个基因区段的6条特异引物,在等温条件下进行核酸扩增反应。对51份实验感染动物及病毒培养标本的H5N1亚型禽流感病毒的HA、NA基因区进行了RT-LAMP检测,并以SYBR Green I为反应指示剂进行了逆转录环介导等温核酸扩增技术,对该反应进行实时监控,经对扩增产物做内切酶验证和测序分析,证明RT-LAMP技术的特异性;同时,用10倍系列稀释的RNA样品对该检测方法的灵敏度进行了测试。结果显示:利用RT-LAMP技术成功检测到H5N1禽流感病毒的HA、NA基因区,且RT-LAMP与Real-time PCR结果呈现很好的一致性。此方法的灵敏度可达到能检测10个拷贝RNA分子水平。因此,RT-LAMP技术应用于H5N1亚型禽流感病毒的快速检测是一种可行的方法。

A simple and sensitive Reverse Transcription Loop-mediated Isothermal Amplification （RT-LAMP） method was established to provide a new alternative for clinical diagnosis of Avian influenza A HSN1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the target for amplification of nucleic acid under isothermal conditions. In current study, fifty- one experimentally infected animal specimens and viral cultures that had been tested were analyzed by RT- LAMP for NA gene and HA gene, respectively. The amplification process of LAMP was monitored in real-time by the addition of SYBR Green dye. Meanwhile, the result showed high correlation between nested PCR and RT-LAMP. The specificity of the RT-LAMP assay was confirmed by restriction enzyme digestion analysis and sequencing of the amplified product. When the sensitivity of this assay was tested by serial 10- fold dilutions of RNA molecules from specimens, it was found that the RT-LAMP method achieved theoretically a sensitivity of 10 RNA molecules. Thus, we concluded that the RT-LAMP assay has potential usefulness for rapid detection of the Avian influenza A HSN1 virus.