摘要
猪非典型瘟病毒(APPV)、经典猪瘟病毒(CSFV)、日本乙型脑炎病毒(JEV)均属于黄病毒科重要成员,从临床症状难以区分且三者的混合感染。为建立一种能快速鉴别检测APPV、CSFV、JEV的三重荧光定量PCR(qPCR)检测方法,本研究根据APPV 5'UTR基因、CSFV E2基因和JEV E基因分别设计引物与探针,将公司合成的重组质粒标准品pLKO.1-APPV-5'UTR、pLKO.1-CSFV-E2和pLKO.1-JEV-E按体积比1:1:1混合后均为(1×10^(8)拷贝/μL)作为模板,经反应条件优化,初步建立同时检测这3种病毒的三重q PCR方法。以本研究构建的APPV慢病毒、CSFV慢病毒、JEV慢病毒,以及猪流感病毒、猪Delta冠状病毒、猪流行性腹泻病毒、伪狂犬病病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒、猪轮状病毒、猪传染性胃肠炎病毒、诺如病毒的基因组为模板,采用建立的该方法检测,评估其特异性,结果显示,该方法仅能检测到APPV、CSFV、JEV的慢病毒,而其他病原检测结果均为阴性。选取1×10^(1)拷贝/μL~1×10^(8)拷贝/μL的混合质粒标准品和1×10^(1)TU/m L~1×10^(6)TU/m L的三种混合慢病毒液分别作为模板,采用本研究建立的方法检测,评估该方法的敏感性,结果显示,该方法对重组质粒pLKO.1-APPV-5'UTR、pLKO.1-CSFV-E2和pLKO.1-JEV-E的检测限均为1×10^(2)拷贝/μL,对3种慢病毒的检测限均为1×10^(2)TU/mL。以1×10~7拷贝/μL、1×10^(5)拷贝/μL、1×10~3拷贝/μL的质粒标准品混合物作为模板,分别进行组内和组间的重复性试验,结果显示,组内和组间重复性试验的变异系数(CV)均低于4.5%。利用本研究建立的三重qPCR和已发表的3种病原的SYBR Green qPCR检测方法对111份临床样品(75份猪肛拭子样品和36份猪组织病料样品)分别检测,结果显示,该三重qPCR方法与已发表的APPV、CSFV、JEV SYBR Green qPCR方法的总符合率分别为99.10%、100%、100%。本研究首次建立的检测APPV、CSFV、JEV的三重TaqMan qPCR方法特异性强、敏感性高、重复性好,为三者临床样品的快速鉴别检测提供了技术支持。
Atypical porcine pestivirus(APPV),classical swine fever virus(CSFV)and Japanese encephalitis virus(JEV)are important members of the Flaviviridae family.They are difficult to distinguish in clinical symptoms and are frequently detected in co-infections.In order to establish a triplex fluorescence quantitative PCR(qPCR)assay for the rapid differential detection of APPV,CSFV and JEV,primers and probes were designed according to the APPV 5'UTR gene,the CSFV E2 gene and the JEV E gene,respectively.The three recombinant plasmid standards pLKO.1-APPV-5'UTR,pLKO.1-CSFV-E2,and pLKO.1-JEV-E synthesized by company were diluted to the same concentration of 1×10^(8)copies/μL and mixed at a volume ratio of 111 as templates,and after optimization of the reaction conditions,a triplex qPCR method for the simultaneous detection of these three viruses was initially established.The specificity of the method was evaluated with the genomes of APPV lentivirus,CSFV lentivirus,JEV lentivirus,SIV,PDCoV,PEDV,PRV,PCV2,PRRSV,PRV,TGEV and NOV as templates.The results showed that the method was positive for detecting APPV lentivirus,CSFV lentivirus,and JEV lentivirus,and negative for detecting SIV,PDCoV,PEDV,PRV,PCV2,PRRSV,PRV,TGEV and NOV.Sensitivity tests were carried out on the mixed standard plasmids of 1×10^(1)copies/μL-1×10^(8)copies/μL and the mixed lentiviruses of 10^(1)TU/mL-10^(6)TU/mL.The results confirmed that that the detection limits of pLKO.1-APPV-5'UTR,pLKO.1-CSFV-E2,and pLKO.1-JEV-E were 1×10^(2)copies/μL,and the detection limits for the three lentiviruses were about 10^(2)TU/mL.The plasmids standard mixture with 1×107 copies/μL,1×10^(5)copies/μL,and 1×103 copies/μL was used as a template for intra-batch and inter-batch repeatability test,and the results showed that the coefficient of variation(CV)of intrabatch and inter-batch repeatability assay were lower than 4.5%.The established triplex qPCR in this study and the published SYBR Green qPCR methods for the three pathogens were used to detect 111 clinical samples(75 pig analswab samples and 36 pig tissue samples).The results showed that the total positive coincidence rates for APPV,CSFV,and JEV were 99.10%,100%,and 100%,respectively.The triplex TaqMan qPCR method established for the first time in this study has strong specificity,highsensitivity,and good reproducibility,which provides technical support for the rapid identification and detection of APPV,CSFV,and JEV.
作者
许浩文
陈军光
马琼琼
潘杏明
郭美君
朱伟群
周莹珊
董婉玉
王晓杜
XU Hao-wen;CHEN Jun-guang;MA Qiong-qiong;PAN Xing-ming;GUO Mei-jun;ZHU Wei-qun;ZHOU Ying-shan;DONG Wan-yu;WANG Xiao-du(China-Australia Joint Laboratory for Animal Health Big Data Analytics,Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management,Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics&Advanced Technology,Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,College of Animal Science and Technology&College of Veterinary Medicine,Hangzhou 311300,China;Ninghai County Animal Health Supervision Institute,Ningbo 315600,China;Ningbo Green Port Aquatic Animal Husbandry Co.,Ltd.,Ningbo 315600,China)
出处
《中国预防兽医学报》
北大核心
2025年第2期144-151,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
浙江省自然科学基金项目(LY23C180003)
浙江省“尖兵·领雁”计划项目(2023C02022、2023C02023)
宁波市公益性科技计划项目(2022S009、2023S103)
杭州市农业产业技术专家团队项目(202306TD17)。
关键词
猪非典型瘟病毒
经典猪瘟病毒
日本乙型脑炎病毒
三重荧光定量PCR
atypical porcine pestivirus
classical swine fever virus
Japanese encephalitis virus
triplex fluorescence quantitative PCR
