新型鸭呼肠孤病毒TaqMan探针实时荧光定量RT-PCR检测方法的建立与应用  

Establishment and application of a TaqMan probe real-time fluorescence quantitative RT-PCR method for the detection of novel duck reovirus

作　　者：张博[1] 陈立功[1,2] 武华 阴雅洁 孟利佳 郭康康 侯绍华 董世山[1,2] ZHANG Bo;CHEN Ligong;WU Hua;YIN Yajie;MENG Lijia;GUO Kangkang;HOU Shaohua;DONG Shishan(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China;Hebei Veterinary Biotechnology Innovation Center,Baoding 071001,China;Beijing Institute of Animal Husbandry and Veterinary Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省兽医生物技术创新中心,河北保定071001 [3]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《河北农业大学学报》2020年第5期80-84,共5页Journal of Hebei Agricultural University

基　　金：国家重点研发计划资助项目(2017YFD0500806);河北省重点研发计划项目(20326622D);河北省创新能力提升计划项目(20567668H)。

摘　　要：为了快速、准确检测新型鸭呼肠孤病毒(Novel duck reovirus,NDRV),本试验采用NDRVσB基因的序列保守区,设计特异性引物和TaqMan探针,经优化反应条件,建立了NDRV实时荧光定量RT-PCR检测方法标准曲线,并进行特异性试验、敏感性试验、重复性试验和临床样品检测试验。结果表明:所建立的TaqMan荧光定量RT-PCR检测方法标准曲线方程为y=-3.354x+44.818,扩增效率为98.7%,标准曲线的相关系数为0.999;该方法可以特异性检测NDRV,但对MDRV、ARV、TMUV、DHAV均无反应信号;该方法检测质粒标准品最低检测浓度为10拷贝/μL,是普通PCR方法检测敏感性的1000倍;该方法的组内与组间变异系数均小于2%;用该方法检测临床样品,结果显示NDRV阳性率为45%,而常规RT-PCR阳性率为33%,比常规RT-PCR敏感。说明本研究建立的TaqMan荧光定量RT-PCR检测方法可为NDRV的监测和早期诊断提供可靠的技术支撑。In order to detect Novel duck reovirus(NDRV)rapidly and accurately,we designed specific primers and TaqMan probes to detect the NDRVσB gene,and optimized the reaction conditions.The test was conducted in the following ways:sensitivity testing,repeatability testing and clinical sample testing.The results showed that the standard curve equation of the TaqMan fluorescence quantitative RT-PCR method was y=-3.354 x+44.818,the amplification efficiency was 98.7%,and the correlation coefficient of the standard curve was 0.999;the method could specifically detect NDRV,but had no reaction signal for MDRV,ARV,TMUV and DHAV;the method detected plasmid standards.The lowest detection concentration was 10 copies/μL,which was 1,000 times more sensitive than the conventional PCR method;the intra-and inter-group coefficients of variation of this method were both less than 2%;the results showed that the positive rate of NDRV was 45%,while the positive rate of conventional RT-PCR was 33%,which was more sensitive than the conventional RT-PCR.The results show that the TaqMan fluorescence quantitative RT-PCR assay established in this study can provide reliable technical support for the monitoring and early diagnosis of NDRV.

关 键 词：新型鸭呼肠孤病毒 σB基因 荧光定量 RT-PCR TAQMAN探针 应用 

分 类 号：S852[农业科学—基础兽医学]