猪细小病毒TaqMan实时荧光定量PCR方法的建立与应用  

Establishment and application of TaqMan real-time quantitative fluorescence method for porcine parvovirus

作　　者：帅文娜 郭子强 李佳乐 罗梦 李丽薇[1,2] 周艳君 姜一峰[1,2] 童武 童光志[1,2] 李兆龙 高飞[1,2,4] SHUAI Wenna;CUO Ziqiang;LI Jiale;LUO Meng;LI Liwei;ZHOU Yanjun;JIANG Yifeng;TONG Wu;TONG Guangzhi;LI Zhaolong;GAO Fei(Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China;Jiangsu Collaborative Innovation Center for the Prevention and Control of Important Animal Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China;College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Shanghai Key Laboratory of Veterinary Biotechnology,Shanghai 200240,China;Institute of A nimal Husbandry and Veterinary Medicine,Fujian Academy of A gricultural Sciences,Fuzhou 350013,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [3]广西大学动物科学技术学院,广西南宁530004 [4]上海市兽医生物技术重点实验室,上海200240 [5]福建省农业科学院畜牧兽医研究所,福建福州350013

出　　处：《中国兽医科学》2025年第1期80-85,共6页Chinese Veterinary Science

基　　金：国家重点研发计划资助项目(2022YFD1800800,2021YFD1801401);上海市自然科学基金资助项目(21ZR1476-900);国家自然科学基金资助项目(32373050,32072861);中国农业科学院科技创新工程资助项目(CAASCS-LPDCP-202402);上海市兽医生物技术重点实验室开放基金资助项目(BD1500010)。

摘　　要：本研究通过对猪细小病毒(PPV)1型非结构蛋白NS1基因保守序列进行设计,建立了PPV的TaqMan实时荧光定量PCR方法,实现了对PPV1的快速检测。结果显示,该检测方法的Ct值与标准品质粒在1.0×10^(10)copies/μL~1.0×10^(4)copies/μL范围内具有良好的线性关系,曲线回归方程为y=-3.6186x+42.01,R^(2)=0.99,扩增效率为189%;重复性好,组内与组间变异系数均低于1.0898%;敏感性好,最低检测限为1.0×10^(1)copies/μL;特异性强,不与猪圆环病毒2型、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪传染性胃肠炎病毒、非洲猪瘟病毒发生交叉反应,并用该方法对临床样品与病毒拷贝数进行了检测。表明本研究建立的方法重复性好、敏感性高、特异性强,适用于对PPV进行临床检测与诊断。In this study,a Taq Man real-time PCR method for PPV was established by designing the conserved sequence of the non-structural protein NS1 gene of porcine parvovirus(PPV)to achieve rapid detection of PPV.The results showed that the Ctvalue of the detection method had a good linear relationship with the standard plasmid at 1.0×10^(10)copies/μL—1.0×10^(4)copies/μL,and the regression equation of the curve was y=-3.6186x+42.01,R^(2)=0.99,and the amplification efficiency was 189%.The reproducibility was good,and the coefficient of variation within and between groups was less than1.0898%.Good sensitivity,the lowest detection limit is 1×10^(1)copies/μL.It has strong specificity and does not cross-react with porcine circovirus type 2,porcine reproductive and respiratory syndrome virus,porcine pseudorabies virus,porcine infectious gastroenteritis virus,African swine fever virus,and the clinical samples and virus copy number were detected by this method.The method established in this study has good reproducibility,high sensitivity and strong specificity,and is suitable for clinical detection and diagnosis of PPV.

关 键 词：猪细小病毒 NS1蛋白 TaqMan实时荧光定量PCR 

分 类 号：S852.659.2[农业科学—基础兽医学]