猪繁殖与呼吸综合征类NADC30 PRRSV荧光定量RT-PCR检测方法的建立及应用  

Establishment and application ofreal-time quantitative RT-PCR method for detecting NADC30-like PRRSV

作　　者：魏春华[1,2,3] 卢贵平 徐叶 刘辰 杨圆 何乐 戴爱玲 杨小燕[1,2,3] 刘建奎 WEI Chunhua;LU Guiping;XU Ye;LIU Chen;YANG Yuan;HE Le;DAI Ailing;YANG Xiaoyan;LIU Jiankui(College of Life Sciences of Longyan University,Longyan,Fujian 364000,China;Fujian Provincial Key Laboratory for the Prevention and Control of Animal Infectious Diseases and Biotechnology,Longyan,Fujian 364000,China;Engineering Research Center for the Prevention and Control of Animal Original Zoonosis,Fujian Province University,College of Life Science,Longyan University,Longyan,Fujian 364012,Fujian,China;College of Animal Science,Fujian Agriculture and Forestry University,Fuzhou,350002,China)

机构地区：[1]龙岩学院生命科学学院,福建龙岩364000 [2]福建省家畜传染病防治与生物技术重点实验室,福建龙岩364000 [3]动物源性人兽共患病防控福建省高校工程研究中心,福建龙岩364000 [4]福建农林大学动物科技学院,福建福州350002

出　　处：《中国兽医学报》2023年第7期1366-1372,共7页Chinese Journal of Veterinary Science

基　　金：中央引导地方科技发展专项资助项目(2021L3028);福建省科技重大专项资助项目(2019NZ09005);福建省科技厅引导资助项目(2021N0032);奇迈基金资助项目(2020SHQM07);大学生创新创业训练资助项目(S202211312024);动物源性人兽共患病防控福建省高校工程中心开放基金资助(2021ZN003)。

摘　　要：为快速检测美洲型猪繁殖与呼吸综合征病毒(PRRSV-2)并从中鉴别诊断NADC30-like PRRSV,根据PRRSV-2的ORF6基因及类NADC30的2个靶点Nsp9和ORF5基因片段分别设计3对特异性引物和探针,建立了一种三重荧光定量RT-PCR检测方法。结果显示,Ct值与标准品在1×10^(9)-1×10^(1)拷贝/μL范围内存在良好的线性关系,R2均>0.990,最低检测浓度为10拷贝/μL;该方法特异性强,与其他基因亚型的PRRSV和其他主要猪源病毒无交叉反应;重复性分析结果显示,组内、组间变异系数均小于1%,呈现出良好重复性。应用建立的三重荧光定量RT-PCR检测方法对116份NADC30-like PRRSV样品进行检测,符合率为98.28%,对458份临床样品进行检测,2对引物单独使用时对NADC30-like PRRSV的检出率分别为36.03%(165/458)和32.3%(148/458),而2对引物同时使用时,NADC30-like PRRSV的检出率明显提高,为44.5%(204/458)。因此,本研究建立的三重荧光定量RT-PCR检测方法可用于PRRSV-2检测,可快速区分出NADC30-like PRRSV,可为PRRSV的防控提供技术支持。In order to establish a multiplex real-time RT-PCR assay for rapid differential detection of PRRSV-2 and NADC30-like strains,specific primers and TaqMan fluorescent probes were designed based on the Nsp9 and ORF5 gene sequence of the NADC30-like PRRSV strains,and special primers and probes based on the relatively conserved sequence of ORF6 of PRRSV-2 were designed.The specificity,sensitivity and reproducibility of the method were evaluated.The results showed that the method had a good linear relationship in the template concentration range of 1×10^(9) copies/μL to 1×10^(1) copies/μL and the correlation coefficient were 0.990,and the minimum detectable concentration was 10 copies/μL.There was no cross reaction with other major swine disease pathogens.The variation coefficient of repeatability test was below 1%,indicating the method had good repeatability.The coincidence rate was 98.28%(114/116)between NADC30-like realtime RT-PCR assay and PRRSV nucleotide sequencing.A total of 458 clinical samples were tested by the real-time RT-PCR,and the positive detection rate of PRRSV-2 and NADC30-like PRRSV were 67.47%(309/458)and 44.5%(204/458),respectively.In contrast,only the primer and probes of ORF5or NSP9was used in real-time RT-PCR,the positive detection rate of NADC30-like PRRSV were 36.03%(165/458)and 32.3%(148/4580),respectively.Therefore,the triple fluorescence quantitative RT-PCR detection method established in this study can be used for PRRSV-2 detection,it can quickly distinguish NADC30-like PRRSV,which will provide technical support for the prevention and control of PRRSV.

关 键 词：猪繁殖与呼吸综合征病毒 TaqMan荧光定量RT-PCR NADC30-like PRRSV 鉴别检测 

分 类 号：S852.65[农业科学—基础兽医学]