猪流行性腹泻病毒通用实时荧光定量RT-PCR方法的建立与初步应用  

Development and application of a universal fluorescent quantitative RT-PCR for detection of porcine epidemic diarrhea virus

作　　者：李玮卓 林青青 刘萍 李军蓝 李静 李娟 成晶 陈振海 陈蕾 骆焕东 LI Weizhuo;LIN Qingqing;LIU Ping;LI Junlan;LI Jing;LI Juan;CHENG Jing;CHEN Zhenhai;CHEN Lei;LUO Huandong(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China;Taizhou LeilingBio Technology Co.,Ltd.,Taizhou 225300,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]扬州大学动物科学与技术学院,江苏扬州225009 [3]泰州蕾灵百奥生物科技有限公司,江苏泰州225300

出　　处：《中国兽医科学》2024年第11期1451-1457,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(32102680);江苏省科学技术厅-基础研究计划(自然科学基金)项目(BK20200931)。

摘　　要：以猪流行性腹泻病毒(PEDV)一段保守的M基因序列为靶标,建立可匹配96.93%现有毒株的PEDV实时荧光定量RT-PCR检测方法。优化反应条件后绘制标准曲线,相关系数R2=0.9995,线性关系良好。该方法与猪繁殖与呼吸综合征病毒、猪轮状病毒、猪链球菌、猪圆环病毒、猪细小病毒、伪狂犬病毒、猪传染性胃肠炎病毒、猪丁型冠状病毒无交叉反应,特异性良好。灵敏度和重复性试验结果显示对PEDV的最低检测限为2.10 copies/μL,且批内、批间重复试验的变异系数均小于2%。用该方法与本病原行业标准方法对162份临床样本进行同批对比检测,结果与行业标准方法的阳性符合率为100%。结果表明,本方法特异性、灵敏度、重复性好,能够有效检测病毒GⅠ和GⅡ基因型,能对导致猪群腹泻的PEDV进行早期确诊。Our study introduces a fluorescent quantitative RT-PCR assay targeting the conserved M gene of porcine epidemic diarrhea virus(PEDV),with the sequences of primers and probes sharing 100%similarities with 96.93%of PEDV strains available including the sequences from Gen Bank and the ones in local databases.After optimizing the reaction conditions,the standard curve was established,and the correlation coefficient(R2)was 0.9995,indicating a good linear relationship.There was no cross-reaction with porcine reproductive and respiratory syndrome virus,porcine rotavirus,Streptococcus suis,porcine circovirus,porcine parvovirus,pseudorabies virus,transmissible gastroenteritis virus of pigs,porcine deltacoronavirus,indicating a good specificity.The limit of detection was determined to be 2.10 copies/μL,with both intra-and inter-batch variations maintaining a coefficient of variation under 2%.Comparative analysis of 162 clinical samples against quarantine protocols revealed a100%positive coincidence rate,underscoring the assay's high sensitivity,specificity,and reproducibility.This advanced fluorescent quantitative RT-PCR method not only effectively identifies both GⅠand GⅡgenogroups of PEDV but also significantly contributes to the early detection and control of the disease,offering a potent tool in virus identification and disease prevention.

关 键 词：猪流行性腹泻病毒 M基因 实时荧光定量RT-PCR TAQMAN探针 

分 类 号：S852.659.6[农业科学—基础兽医学]