猪流感病毒一步法荧光定量RT-PCR诊断方法的建立

Development of one-step real-time quantitative RT-PCR approach for the detection of swine influenza virus

为了建立一种在猪流感流行病学调查和临床诊断中应用的快速高效的诊断方法,根据猪流感病毒(swine influenza virus,SIV)M基因的保守序列,设计并合成1对特异性引物和1条特异性TaqMan探针,建立了以RNA为反应模板的一步法荧光定量RT-PCR诊断方法。使用含有M基因的重组质粒作为标准品绘制标准曲线,该曲线效率值为2.014,误差值为0.005。试验结果表明,该方法灵敏度极高,最低可检测到10 copies的核酸;临床样品检测结果也显示其灵敏度高于普通PCR和病毒分离法;特异性强,对猪群常见的其他6种病毒检测结果均为阴性。临床样品检测显示,该方法操作简单,可以用RNA作为模板直接检测,节省时间,而且特异性好、灵敏度高,是可以应用于猪流感流行病学调查的一种可靠的诊断方法。

In order to develop a rapid and high-performance assay method for epidemiological study and clinical diagnosis of swine influenza virus,a one-step real-time quantitative RT-PCR method based on a TaqMan probe was developed.Conserved regions in the M gene of swine influenza viruses served as targets for primers and TaqMan probe.Concentrations of primers and probe were optimized to improve the sensitivity and specificity of reactions.A series of dilutions of recombinant plasmid were prepared and used to generate standard curve with an efficiency value of 2.014 and a error value of 0.005.Results showed that the threshold of 10 copies of target molecules can be detected,which revealed high sensitivity of the method.Clinical diagnosis showed that this approach was more sensitive than normal RT-PCR and virus isolation methods.Besides,it was negative for another six popular viruses in swine,which showed high specify of the approach,Moreover,it was easy to be operated and was time-saving.In conclusion,the method established in the present study can be used for rapid detection and monitoring in epidemiology study of SIV.