传染性法氏囊病病毒和禽致病性大肠杆菌的二重探针实时荧光定量PCR方法的建立与应用  

Establishment and Application on a Duplex FQ-PCR for Detection of Infectious Bursal Disease Virus and Pathogenic Escherichia coli

作　　者：陈金南 陈睿 李尚泉 李静玲 陈容慧 农芳婷 王威威 李宜海 王林果 韦平 何秀苗 CHEN Jinnan;CHEN Rui;LI Shangquan;LI Jingling;CHEN Ronghui;NONG Fangting;WANG Weiwei;LI Yihai;WANG Linguo;WEI Ping;HE Xiumiao(College of Marine and Biotechnology/Guangxi Key Laboratory of Polysaccharide Materials and Modification,Guangxi Minzu University,Nanning,Guangxi 530006;Institute for Poultry Science and Health,Guangxi University,Nanning,Guangxi 530005;Pulike Biological Engineering Inc.,Luoyang,Henan 471000)

机构地区：[1]广西民族大学海洋与生物技术学院,广西多糖材料与改性重点实验室,广西南宁530006 [2]广西大学养禽与禽病学研究所,广西南宁530005 [3]普莱柯生物工程股份有限公司,河南洛阳471000

出　　处：《中国家禽》2023年第9期31-37,共7页China Poultry

基　　金：国家自然科学基金项目(32160824);国家现代农业产业技术体系广西肉鸡产业创新团队建设专项(nycytxgxcxtd-19-03);广西研究生教育创新计划资助项目(YCBZ2022034)。

摘　　要：为建立用于传染性法氏囊病病毒(IBDV)和禽致病性大肠杆菌混合感染快速鉴别检测的实时荧光定量PCR(FQ-PCR)方法,研究分别根据IBDV VP2基因和大肠杆菌UidA基因设计合成特异性引物和探针,通过普通PCR方法扩增目的基因片段并克隆至pGM-T载体,构建重组质粒pGM-T/VP2和pGM-T/UidA作为标准品,通过正交试验确定引物和探针的最佳浓度,分别建立基于VP2和UidA基因的标准扩增曲线,并进一步验证其特异性、灵敏性、重复性,建立的方法应用于人工感染样本和临床样本的检测,并与常规PCR方法进行比较。结果显示:VP2和UidA基因的最佳上、下游引物浓度均为0.30μmol/L,VP2和UidA基因的探针浓度分别为0.35μmol/L和0.25μmol/L;VP2和UidA基因扩增的线性范围均为10^(3)~10^(7)拷贝/μL,相关系数R2≥0.99,灵敏度均为100拷贝/μL;两者组间及组内变异系数均小于2%;人工感染样品与临床样品的检测结果与普通PCR方法一致。研究表明,建立的二重探针FQ-PCR方法为同时检测IBDV和禽致病性大肠杆菌提供了快速、敏感、特异且能满足临床样本需求的检测方法。In order to establish a duplex real-time fluorescence quantitative PCR(FQ-PCR)to detect infectious bursal disease virus(IBDV)and avian pathogenic Escherichia coli,the specific primers and probes were designed and synthesized according to VP2 gene of IBDV and UidA gene of Escherichia coli(E.coli),respectively.Then VP2 gene and UidA gene were amplified by common PCR and cloned into pGM-T vector to construct recombinant plasmids pGM-T/VP2 and pGM-T/UidA,which were used as standard sample.Based on the standard plasmid,the optimal concentration of primers and probes were determined by orthogonal test and the standard amplification curves of VP2 and UidA gene were established.The specificity,sensitivity and repeatibility of the amplification curves were further verified,and the established duplex FQ-PCR was applied to the detection of artificial infection samples and clinical samples compared with the common PCR.The results showed that the optimal concentrations of primers for both VP2 and UidA were 0.30μmol/L.The optimal concentrations of probes for VP2 and UidA was 0.35μmol/L and 0.25μmol/L,respectively.The linear range of VP2 and UidA gene was 103 to 107 copies/μL,the correlation coefficient R2 was equal or greater than 0.99,the sensitivity was 100 copies/μL,the coefficient of variation between and within groups was less than 2%.The results of artificial infection samples and clinical samples detected by the duplex FQ-PCR assay were consistent with that of the common PCR.The results indicated that the established duplex FQ-PCR could provide a rapid,sensitive and specific detection method for the simultaneous detection of IBDV and avian pathogenic E.coli.

关 键 词：IBDV 禽致病性大肠杆菌 混合感染 二重FQ-PCR 

分 类 号：S855.1[农业科学—临床兽医学] S855.3[农业科学—兽医学]