新型鸭呼肠孤病毒一步法TaqMan-MGB荧光定量RT-PCR检测方法的建立与应用

Development and application of a one-step real-time TaqMan-MGB RT-PCR assay for detection of novel duck reovirus

为建立一种快速、敏感和特异性检测新型鸭呼肠孤病毒(novel duck reovirus,NDRV)的方法,本研究根据GenBank数据库中NDRV S3基因保守序列设计1对特异性引物和1条MGB荧光探针,建立了一种检测NDRV的一步法MGB荧光定量RT-PCR方法,对其特异性、敏感性和重复性进行检验,并与普通RT-PCR进行比较。结果显示,扩增标准曲线相关系数为0.999,扩增产物的熔解曲线仅出现单特异峰。该方法对经典鸭呼肠孤病毒、H9N2禽流感病毒、鸭坦布苏病毒、A型鸭肝炎病毒、鸭新城疫病毒、鸭瘟病毒及番鸭细小病毒均未检测到信号,对NDRV的最小检出量为10拷贝·μL^-1。组内和组间变异系数均小于2%,重复性好。此外,利用该方法对239份疑似新型呼肠孤病毒病样品进行检测,常规RT-PCR检测时有75份为阳性,一步法TaqMan-MGB荧光定量RT-PCR方法检测时有100份为阳性,而且常规RT-PCR检测出的阳性样品用一步法TaqMan-MGB荧光定量RT-PCR检测时均为阳性,符合率为100%。该检测方法的建立为NDRV早期快速检测及定量分析提供新的方法。

To develop a fast,sensitive,specific TaqMan MGB probe real-time fluorescent quantitative PCR assay for detecting novel duck reovirus(NDRV),a pair of specific primers and a MGB probe were designed according to the conserved region of S3 segment of the NDRV genome.The results showed that recombinant plasmids were used to generate standard curve with correlation coefficient(R2)of 0.999,and the melting curve showed one specific peak.No cross-reaction was detected for classical muscovy duck reovirus(CDRV),avian influenza virus(AIV),duck Temubusu virus(DTMUV),duck hepatitis A virus(DHV-1),Newcastle diseases virus(NDV),duck plague virus(DPV)and muscovy duck parvovirus(MDPV).The limit detection of real-time RT-PCR was about 10 copies·μL^-1 for the target gene.The reproducibility tests in inter-assay and intra-assay indicated that the coefficients of variation were less than 2%.In addition,a collection of 239 clinical samples from Zhejiang during the year 2011-2015 were detected by the assay and conventional RT-PCR,respectively.The results showed that 75 samples were positive in the conventional RT-PCR test while 100 samples were positive in the one-step MGB probe real-time RT-PCR test.All the positive samples detected by conventional RT-PCR were positive by one-step MGB probe RT-PCR test,and the coincidence rate was 100%.The method established in this study could be useful for earlier rapid laboratory diagnosis and quantitative analysis of the infection of NDRV.