H5亚型禽流感病毒实时荧光RT-PCR方法的建立与应用

Establishment and Application of Real-time RT-PCR for Detection of H5 Subtype Avian Influenza Virus

H5亚型禽流感病毒HA序列差异越来越大。为更准确地检测H5亚型禽流感病毒,对GenBank中发表的H5亚型禽流感病毒HA序列以及本实验室保存病毒序列进行比对,同时设计1对引物和1条探针,建立了H5亚型禽流感病毒实时RT-PCR方法,并对该方法的反应体系和反应参数进行了优化。以本实验室分离测序确定的30份H5亚型禽流感病毒RNA为模板,将该方法与两种商品化H5亚型禽流感病毒检测试剂盒进行比对,发现该方法与两种商品化试剂盒的检出率分别为100%、98%、98%。敏感性试验显示,该方法可以检测出0.1 fg的RNA模板,灵敏度比两种商品化试剂盒均提高了10倍。结果表明,该方法具有更高的特异性和敏感性,可用于H5亚型高致病性禽流感的早期诊断。

In recent years,the difference in HA sequence of H5 subtype avian influenza virus(AIV)was increasing. In order to detect H5 subtype AIV more accurately,by comparing the HA sequence of H5 subtype AIV published in GenBank with the one conserved in the laboratory and designing a pair of primers and one TaqMan probe,a real-time RT-PCR method was developed,and its reaction system and parameters were optimized. Taking 30 RNA samples of H5 subtype AIV strain isolated and sequenced by the laboratory as the template,the established method was compared with two commercial H5 subtype AIV detection kits,and it was found that the detection rates were 100%,98% and 98% respectively. Sensitivity test showed that RNA template of 0.1 fg could be detected by the established method whose sensitivity was 10 times higher than that of the two commercial kits. In conclusion,the RT-PCR method was more specific and sensitive,and it could be used for early diagnosis of H5 subtype highly pathogenic avian influenza.