猪腹泻病毒一步法多重TaqMan荧光定量RT-PCR检测法的建立及应用  

One-Step Multiple Taq Man Real-time RT-PCR for Simultaneous Detection of Swine Diarrhea Viruses

作　　者：王一丹 杨发龙[1] 陈弟诗[2] 向华 任玉鹏 WANG YiDan;YANG FaLong;CHEN DiShi;XIANG Hua;REN YuPeng(College of Animal Science and Veterinary Medicine,Southwest Minzu University,Chengdu 610041;Sichuan Provincial Center for Animal Disease Prevention and Control,Chengdu 610041)

机构地区：[1]西南民族大学畜牧兽医学院,成都610041 [2]四川省动物疫病预防控制中心,成都610041

出　　处：《中国农业科学》2023年第1期179-192,共14页Scientia Agricultura Sinica

基　　金：中央高校基本科研业务费专项资金(2020NQN29)。

摘　　要：【目的】建立一种可同时检测猪流行性腹泻病毒(PEDV)野毒株、猪A群轮状病毒(GARV)、猪德尔塔冠状病毒(PDCoV)、猪阿尔法冠状病毒(SADS-CoV)及猪捷申病毒(PTV)5种猪腹泻病毒的一步法多重TaqMan荧光定量RT-PCR检测法。为猪腹泻病的快速诊断和流行病学调查提供高效灵敏的工具。【方法】对PEDV多个基因型毒株ORF3基因比对分析,以PEDV野毒株为模板,对疫苗株ORF3基因稳定缺失区域设计特异性探针,并在两端保守区域设计上下游引物;在靠近GARV G3、G4、G5和G9型NSP5基因5′端保守碱基区域设计引物及探针,并加入简并碱基。同时,分别选择PDCoV M基因、PTV 5′UTR序列、SADS-CoV N基因等保守基因设计特异性引物及探针,用于多重荧光定量PCR方法的建立。对引物、探针浓度和退火温度进行优化;用RStudio参照代码绘制ROC曲线,确定检测方法的敏感度值、特异度值及曲线下面积AUC,并计算Youden指数,最终确定检测临界值;从阳性核酸中扩增靶基因,并克隆至pEASY-T1载体。通过体外转录,获得5种标准品分别命名为:cRNA-PEDV、cRNA-GARV、cRNA-PDCoV、cRNA-PTV和cRNA-SADS-CoV。对检测方法的敏感性、特异性和重复性等进行评估;并与同类方法对临床样本的检测符合率进行比较。【结果】得到了5种病原检测的最佳引物、探针浓度和最佳退火温度。根据ROC曲线确定PEDV、GARV、PDCoV、PTV和SADS-CoV临界CT值分别为:35.78、34.25、34.98、34.60和35.70;5种病原的检测下限均可达到1×10^(2) copies/μL,标准曲线线性关系良好,扩增效率在96.3%-104%之间;该方法对PEDV CV777疫苗株、PEDV AJ1102疫苗株、猪传染性胃肠炎病毒(TGEV)、猪瘟病毒(CSFV)、猪伪狂犬病毒(PRV)、猪繁殖与呼吸障碍综合征病毒(PRRSV)、猪霍乱沙门氏菌(S.choleraesuis)、多杀性巴氏杆菌(P.multocida)、大肠杆菌(E.coli)、猪链球菌(S.suis)和葡萄球菌(S.aureus)等多种菌毒株均不检出,具【Objective】 The aim of this study was to establish a one-step multiplex real-time RT-PCR method to simultaneously detect and quantify five swine diarrhea related viruses, PEDV, GARV, PDCoV, SADS-CoV and PTV, so as to provide an efficient and sensitive tool for rapid diagnosis and epidemiological investigation of porcine diarrhea.【Method】The ORF3 gene sequences of several genotypes of PEDV were analyzed, and then the primers and probes were designed for detection of PEDV field strains by referring to the ORF3 genes, which contained deletion mutations in attenuated strains. The 5’-end conserved region of NSP5 genes of GARV G3, G4, G5 and G9 strains were analyzed for design of probes and primers. The specific primers and probes targeting to the conserved regions of PDCo V M, PTV 5’UTR and SADS-CoV N genes were designed for detection of the pathogens. The ROC curves were completed by referring to parameters that were set in RStudio. The specificity value, sensitivity value, and areas under the curves(AUC) and Youden value were calculated according to ROC curves to determine the cut-off CT value.The amplified fragments were cloned into pEASY-T1 vector. The standards prepared through in vitro transcription were named as c RNA-PEDV,cRNA-GARV, cRNA-PDCoV, cRNA-PTV and cRNA-SADS-CoV. The sensitivity, specificity and repeatability of one-step multiplex real-time RT-PCR were evaluated. Coincidence rate between this and another similar method were compared in the detection of clinical samples. 【Result】 Both the annealing temperature and optimal concentrations of primers and probes were obtained for detection of the five pathogens. According to the ROC curve, the CT cut off values for detection of PEDV, GARV, PDCoV, PTV, and SADS-CoV were set as 35.78, 34.25, 34.98, 34.60, and 35.70, respectively. The detection sensitivity of this method for the five pathogens could reach 1×10^(2) copies/μL. The standard curves had a good linear relationship and the amplification efficiency was between 96.3% and 104%. The es

关 键 词：一步法多重TaqMan荧光定量RT-PCR 猪腹泻相关病毒 鉴别诊断 检测临界值 

分 类 号：S858.28[农业科学—临床兽医学]