摘要
猪肠道冠状病毒(SeCoV)包括猪流行性腹泻病毒(PEDV)、传染性胃肠炎病毒(TGEV)和猪德尔塔冠状病毒(PDCoV),是引起仔猪严重腹泻的主要病原体,给养猪业造成了巨大的损失。为快速、准确地检测和鉴别这3种病毒,本研究建立了一种三重实时荧光定量反转录PCR(RT-qPCR)方法,用于同时检测PEDV、TGEV和PDCoV。根据PEDV M基因、TGEV ORF 1b基因和PDCoV ORF 1b基因的保守序列分别设计了特异性引物和探针。优化反应条件后,成功建立了一种能同时检测PEDV、TGEV和PDCoV的三重RT-qPCR方法。该方法对这3种病毒具有良好的特异性,与猪场常见的猪瘟病毒(CSFV)、猪圆环病毒2型(PCV2)、猪A群轮状病毒(PoRVA)、猪伪狂犬病毒(PRV)和猪繁殖与呼吸综合征病毒(PRRSV)等均无交叉反应。对重组质粒pTOPO-PEDV 128、pTOPO-TGEV 116和pTOPO-PDCoV 125线性模板的检测极限分别为16.835、17.610、17.020拷贝/μL。组内、组间变异系数均小于5%,且组间差异不显著(P>0.05)。与标准方法的符合性比较结果显示,三重RT-qPCR的检测符合率为100%,在35份组织样本的检测中,17份呈PEDV阳性,阳性率为48.57%(17/35),TGEV和PDCoV的检测结果均为阴性,未检测到混合感染。结果表明,建立的三重RT-qPCR方法特异、灵敏、稳定、快捷,可同时用于PEDV、TGEV和PDCoV的临床检测和鉴别诊断,为猪腹泻冠状病毒的检测和流行病学调查提供了一种高效、可靠的技术手段。
Swine enteric coronaviruses(SeCoV),such as porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),and porcine delta coronavirus(PDCoV),cause severe diarrhea in piglets,resulting in substantial losses in pig farming.In this study we establish a triple fluorescence reverse transcription-quantitative PCR(RT-qPCR)method for the simultaneous de-tection of PEDV,TGEV,and PDCoV.The specific primers and probes for each target virus were designed based on conserved sequences from the PEDV M gene,the TGEV ORF 1b gene,and the PDCoV ORF 1b gene respectively.Following the optimization of parameters and conditions,a triple RT-qPCR method was successfully established to simultaneously detect PEDV,TGEV,and PD-CoV.The developed assay exhibits strong specificity for these three pathogens without any cross-reaction with other common porcine viruses like CSFV,PCV2,PoRVA,PRV,and PRRSV.The de-tection limit of linear templates for pTOPO-PEDV 128,pTOPO-TGEV 116,and pTOPO-PDCoV 125 recombinant plasmids were 16.835,17.610 and 17.020 copies/μL,respectively.The intra group and inter group coefficients of variation were less than 5%,with no significant differences observed(P>0.05).Moreover,the detection consistency rate of the developed RT-qPCR was compared with standard method and showed 100%agreement.Out of 35 small intestine tissue samples,17 tested positive for PEDV,resulting in a positive rate of 48.57%(17/35).The tests for TGEV and PDCoV yielded negative results,and no mixed infections were detected.Based on the above results,the tri-ple RT-qPCR method established is specific,sensitive,stable,and rapid,and can be used for clinical detection and differential diagnosis of PEDV,TGEV,and PDCoV simultaneously,providing a method for the detection and epidemiological investigation of porcine diarrhea coronaviruses.
作者
陈千林
李绍梅
张邑帆
牟豪
刘明妮
杨柳
郭庆勇
付利芝
CHEN Qianlin;LI Shaomei;ZHANG Yifan;MU Hao;LIU Mingni;YANG Liu;GUO Qingyong;FU Lizhi(Disease Prevention and Control Research Institute,National Center of Technology Innovation for Pigs,Rongchang,Chongqing 402460,China;College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;Veterinary and Veterinary Medicine Research Institute,Chongqing Academy of Animal Sciences,Rongchang,Chongqing 402460,China)
出处
《中国兽医学报》
北大核心
2025年第5期905-912,共8页
Chinese Journal of Veterinary Science
基金
重庆英才计划“包干制”资助项目(cstc2022ycjh-bgzxm0183)
国家生猪技术创新中心先导科技资助项目(NCTIP-XD/B11)。
作者简介
陈千林(1991-),男,博士研究生;通信作者:郭庆勇,E-mail:wlmqgqy@126.com;通信作者:付利芝,E-mail:flzfulizhi@163.com。
