Li Qi-Ming, Ma Xue-Jun, Gao Han-Chun, Zhou Rui, Kuang Zhi-Zhou, Hou Yun-De
State Key Laboratory for Molecular Virology and Genetic Engineering, Beijing 100052, China.
Bing Du Xue Bao. 2008 May;24(3):178-84.
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 H5N1 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 H5N1 virus.
建立了一种简单且灵敏的逆转录环介导等温扩增（RT-LAMP）方法，为甲型H5N1禽流感病毒的临床诊断提供了一种新的选择。该方法采用一组六个特别设计的引物，这些引物识别靶标的八个不同序列，以便在等温条件下扩增核酸。在当前研究中，分别通过RT-LAMP对51个经检测的实验感染动物标本和病毒培养物的NA基因和HA基因进行了分析。通过添加SYBR Green染料实时监测LAMP的扩增过程。同时，结果显示巢式PCR与RT-LAMP之间具有高度相关性。通过限制性内切酶消化分析和扩增产物测序证实了RT-LAMP检测的特异性。当通过对标本中的RNA分子进行连续10倍稀释来测试该检测方法的灵敏度时，发现RT-LAMP方法理论上达到了10个RNA分子的灵敏度。因此，我们得出结论，RT-LAMP检测方法在快速检测甲型H5N1禽流感病毒方面具有潜在用途。
