H6亚型禽流感病毒荧光定量RT-PCR检测方法的建立

本研究旨在建立一种快速、特异、敏感的H6亚型禽流感病毒(AIV)实时荧光定量RT-PCR检测方法。针对H6亚型AIV的血凝素(HA)基因序列保守区设计1对引物,并优化反应条件。结果表明,该方法的最低检测限为1.07×101拷贝质粒DNA,与常规PCR相比,灵敏度高出1 000倍;扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,Tm值为(80.81±0.08)℃,组内变异系数为0.08%～0.99%,组间变异系数为1.85%,可重复性好;对H1、H3、H5、H7、H9亚型禽流感病毒及新城疫病毒等其他呼吸道病原体无扩增;检测快速,从样本处理到报告结果仅需3.5 h。

Establishment of Real-time RT-PCR for rapid detection of H6 subtype avian influenza virus

The aim of this study is to develop a rapid,sensitive and specific Real-time RT-PCR(RRT-PCR) method for detection of H6 subtype avian influenza virus(AIV).A pair of primers specific to the hemagglutinin(HA) gene was designed according to the HA gene sequences of H6 subtype AIV available in GenBank,and the reaction conditions were optimized.The results showed that,the detection limit of RRT-PCR was 1.07×101 plasmid copies.The RRT-PCR was more sensitive than conventional PCR by 1000 folds.The melting curve analysis showed one specific peak at the melting temperature of(80.81±0.08)℃ without primer-dimers peak.The reproducibility of RRT-PCR was high and the coefficients of intra-and inter-assay assay variation were 0.08%~0.99% and 1.85%,respectively.The specificity of the assay was high without any amplification for other viruses.In the RRT-PCR assay,the required time from sample processing to reporting of results was only 3.5h.