检测PRRSV及鉴别HP-PRRSV毒株的二重PCR与二重TaqMan荧光定量PCR方法  

Discrimination of porcine reproductive and respiratory syndrome virus(PRRSV) and highly pathogenic PRRSV strain with a duplex PCR and a duplex TaqMan real-time PCR

作　　者：陈旭 汤德元[1] 曾智勇[1] 王彬[1] 袁盛林 廖正波 何松 周飘 毛茵茗 CHEN Xu;TANG Deyuan;ZENG Zhiyong;WANG Bin;YUAN Shenglin;LIAO Zhengbo;HE Song;ZHOU Piao;MAO Yinming(College of Animal Science,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025

出　　处：《中国兽医科学》2024年第3期316-324,共9页Chinese Veterinary Science

基　　金：贵州省科技支撑计划项目(黔科合支撑[2021]一般162)。

摘　　要：为了实现猪繁殖与呼吸综合征病毒(PRRSV)早期检测及快速鉴别高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)变异株,利用HP-PRRSV Nsp2蛋白第481位和532~560位氨基酸缺失的特性设计2对引物,优化反应条件建立二重PCR检测方法,同时针对PRRSV M基因和HP-PRRSV Nsp2基因设计特异性引物和探针,优化反应条件建立二重TaqMan荧光定量PCR方法,并评估这2种方法的敏感性、特异性、重复性。敏感性试验结果显示:二重PCR方法的检测下限分别为0.058 ng/μL(PRRSV)和4.7 ng/μL(HP-PRRSV);二重TaqMan荧光PCR方法的检测下限分别为10 copies/μL(PRRSV)和100 copies/μL(HP-PRRSV)。这2种方法与其他猪病病原均不发生非特异性反应,重复性试验结果良好。经131份临床样品检测,可得到PRRSV的阳性率约12.2%(16/131)、HP-PRRSV的阳性率约17.6%(23/131)、二者的混合感染率约8.4%(11/131)。证实,本研究成功建立了二重PCR和二重TaqMan荧光定量PCR检测方法,可为临床上猪繁殖与呼吸综合征的防控提供技术支持。In order to achieve early detection of porcine reproductive and respiratory syndrome virus(PRRSV) and rapid identification of highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV) variants,two pairs of specific primers were designed based on characteristics of amino acid deletion at positions 481 and 532—560 of HP-PRRSV Nsp2 protein,a duplex PCR detection method was established by optimizing the reaction conditions.The specific primers and probes were designed based on PRRSV M gene and HP-PRRSV Nsp2 gene,a duplex Taq Man real-time PCR method was established by optimizing the reaction conditions.The sensitivity,specificity,and reproducibility of both methods were evaluated.The sensitivity test results showed that the lowest detection limits of the duplex PCR method were 0.058 ng/μL(HP-PRRSV) and 4.7 ng/μL(HP-PRRSV);the minimum detection limits of the duplex Taq Man real-time PCR method were 10 copies/μL(PRRSV)and 100 copies/μL(HP-PRRSV),and there was no cross-reactivity between these two detection methods and other swine pathogens,and the repeatability test results were good. A total of 131 clinical samples detection showed positive rate of PRRSV was 12.2%(16/131),the positive rate of HP-PRRSV was 17.6%(23/131),and mixed infection rate was 8.4%(11/131). It was confirmed that the duplex PCR and duplex Taq Man real-time PCR detection methods were successfully established,which could provide technical support for the prevention and control of porcine reproductive and respiratory syndrome in clinical practice.

关 键 词：猪繁殖与呼吸综合征病毒 高致病性猪繁殖与呼吸综合征病毒 二重PCR 二重TaqMan荧光定量PCR 鉴别检测 

分 类 号：S852.659.6[农业科学—基础兽医学]