禽流感病毒H5亚型RT-exoRPA检测方法的建立

Establishment of RT-exoRPA method for detection of H5 subtype avian influenza virus

为建立一种特异、灵敏、简便、快速的禽流感病毒H5亚型的检测方法,根据近几年流行的禽流感病毒H5亚型毒株HA基因序列,采用DNAStar生物分析软件进行基因序列比对后,选择相对保守区域,设计多对引物及探针,建立了禽流感病毒H5亚型RT-exoRPA检测方法,对该方法的反应条件进行优化并检测特异性和灵敏性。结果显示,建立的检测禽流感病毒H5亚型RT-exoRPA方法特异性强,只有禽流感病毒H5亚型呈阳性,其他均为阴性;灵敏性较高,最低可检测到质量浓度为0.89×10^-3 mg/L的禽流感病毒H5亚型核酸,比实时荧光RT-PCR方法灵敏性低10倍;检测快速,检测反应所需时间仅20min,比实时荧光RT-PCR方法缩短近1h。结果表明,所建立的禽流感病毒H5亚型RT-exoRPA检测方法适应于禽流感病毒H5亚型的现场检测,以及基层实验室的推广应用。

In order to establish a specific,sensitive,simple and rapid H5 subtype avian influenza virus(AIV)detection method,a series of specific primers were designed based on the sequences of H5 subtype avian influenza virus hemagglutinin(HA)gene and analysis of DNAStar bioanalysis software.By optimized conditions,the isothermal reverse transcription recombinase polymerase amplification(RT-exoRPA)for detection of H5 subtype avian influenza virus was established.Specificity and sensitivity of the RT-exoRPA assay were detected.The results showed that the RT-exoRPA method with strong specificity.The detection limit of RT-exoRPA for virus genome DNA was about 0.89×10-3 mg/L,slightly lower than that of real-time RT-PCR.It cost about 20 minutes shorter near 1 hthan the real-time fluorescent RT-PCR method.Thus the RT-exoRPA method for detection of H5 subtype avian influenza virus is suitable for use in field conditions and primary level laboratory.