猪繁殖与呼吸综合征病毒通用微芯片荧光定量RT-PCR方法的建立和初步应用  

Establishment and application of universal microchip real-time RT-PCR assay for detection of porcine reproductive and respiratory syndrome virus

作　　者：陈玉红 徐琦[1] 刘玉良[1] 王传彬[1] 周智[1] 张硕[1] 倪建强[1] 刘洋[1] CHEN Yu-hong;XU Qi;LIU Yu-liang;WANG Chuan-bin;ZHOU Zhi;ZHANG Shuo;NI Jian-qiang;LIU Yang(China Animal Disease Control Center,National/OIE Porcine Reproductive and Respiratory Syndrome Reference Laboratory,Beijing 102618,China;College of Animal Science and Technology,Guangxi University,Nanning 530004,China)

机构地区：[1]中国动物疫病预防控制中心国家/OIE猪繁殖与呼吸综合征参考实验室,北京102618 [2]广西大学动物科学技术学院,广西南宁530004

出　　处：《中国兽医科学》2021年第11期1361-1368,共8页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2018YFD0500800);国家重点研发计划项目(2016YFD0500705)。

摘　　要：为建立一种可用于猪繁殖与呼吸综合征病毒(PRRSV)通用的核酸检测技术,本研究根据已发表的多株PRRSV ORF6(M)基因保守序列,设计特异性引物和探针,通过优化反应体系和条件,成功建立了一种可用于PRRSV通检的微芯片荧光RT-PCR检测方法。结果显示,该方法特异性强,与多种常见的猪病病毒均无交叉反应;通检性好,可以检出包括美洲型、欧洲型、高致病性猪繁殖与呼吸综合征病毒以及NADC30-like等多种基因型PRRSV毒株;灵敏性好,检测下限为1×10^(1)TCID_(50)/mL,与常规荧光RT-PCR方法一致;重复性好,批内和批间重复性变异系数均小于4%;且成本低,模板用量少,反应快速,全程仅需61 min;对80份临床样品的检测结果与常规荧光RT-PCR的符合率为100%。本研究建立的微芯片荧光定量RT-PCR技术为PRRSV的快速、通用型检测提供了有力的技术支撑。In order to establish a method that can detect porcine reproductive and respiratory syndrome virus(PRRSV),in this study,a universal microchip real-time RT-PCR(RT-q PCR)assay was developed based on the conserved nucleotide sequence of ORF 6(M)gene for detection of PRRSV.The results revealed that the approach we developed was highly specific,as no cross reaction with different viruses of common swine diseases was detected.A variety of PRRSV strains,including American type,European type,highly pathogenic(HP)PRRSV and NADC30-like,could be detected by the new method.The detection limit was 1×10^(1) TCID_(50)/m L,showing a sensitivity that was comparable to that of the conventional real-time RT-PCR.Furthermore,the method has good reproducibility,and the coefficient of variation of intra-and inter-batch repeatability was less than 4%.Importantly,the approach has the advantages of low cost,less template consumption,rapid reaction,allowing the entire testing time needed was only approximately 1 h.A total of 80 clinical samples were tested by our method and the result was consistent with that obtained by conventional real-time RT-PCR.In conclusion,the novel approach we established is specific,rapid,universal and is suitable for detection of PRRSV in surveillance and clinical diagnosis.

关 键 词：猪繁殖与呼吸综合征病毒 通用 荧光定量RT-PCR 微芯片 

分 类 号：S852.659.6[农业科学—基础兽医学]