检测鸡传染性支气管炎病毒GVI-1基因型毒株的实时荧光定量PCR方法建立  

Establishment of a RT-qPCR assay for detection of GVI-1 genotype of avian infectious bronchitis virus

作　　者：陈淑琴 杜旭彬 鄢坤 廖凯 张成成 郭梦娇 吴艳涛[1] 张小荣[1] CHEN Shuqin;DU Xubin;YAN Kun;LIAO Kai;ZHANG Chengcheng;GUO Mengjiao;WU Yantao;ZHANG Xiaorong(Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,College of Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225009,China)

机构地区：[1]扬州大学兽医学院江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医学报》2021年第11期2121-2125,2131,共6页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2017YFD0500703);现代农业产业技术体系建设专项基金资助项目(CARS-40-k16);扬州大学“高端人才支持计划”基金资助项目;江苏高校优势学科建设工程基金资助项目。

摘　　要：为快速鉴别GVI-1基因型鸡传染性支气管炎病毒(infectious bronchitis virus,IBV)毒株与其他基因型毒株,根据IBV不同基因型毒株S1基因序列差异较大的区域设计特异性引物和探针,建立了检测GVI-1基因型毒株的实时荧光定量PCR(qPCR)方法。该方法在1.0×10^(1)~1.0×10^(10)拷贝/μL范围内具有良好的线性关系,相关系数(R^(2))为0.999;对质粒标准品的检测下限可达1.0×10^(1)拷贝/μL;与GI-1、GI-7、GI-13、GI-19和GI-28等其他基因型毒株和疫苗株均无交叉反应;组内和组间重复试验的变异系数均小于2.5%;应用该方法和常规PCR方法对人工感染雏鸡的口腔和泄殖腔棉拭子进行检测,2种方法的符合率为96.7%,且qPCR方法敏感性更高。结果表明,本研究建立的GVI-1基因型IBVqPCR检测方法灵敏度高,特异性强,可重复性好,可应用于GVI-1基因型IBV流行病学监测和临床分离株的快速筛查。In order to rapidly distinguish the GVI-1 strains from other genotypes of avian infectious bronchitis virus(IBV),specific primers and probes were designed based on the S1 gene sequence alignment analysis among different genotypes to establish a real-time quantitative PCR(RT-qPCR)assay.The results demonstrated that this assay showed good linearity within the concentration range tested,with a correlation coefficient of 0.999.The limit of detection was 1.0×10^(1) copies/μL.The assay was specific against GVI-1 strains and did not exhibit cross reactivity with other genotype strains such as the GI-1,GI-7,GI-13,GI-19 and GI-28.The variation coefficients of repeated test intra-and inter-group were both less than 2.5%.This assay and PCR were used to detect virus of oral and cloacal swabs collected from infected chicks.The coincidence rate of the two methods was 96.7%,and the sensitivity of RT-qPCR assay was higher.In conclusion,the assay for detection of GVI-1 genotype of IBV established in this study had good specificity,sensitivity,and repeatability,which has important implications for GVI-1 genotype of IBV in epidemiological surveillance and rapid screening for clinical isolates.

关 键 词：鸡传染性支气管炎病毒 GVI-1基因型 实时荧光定量PCR 鉴别检测 

分 类 号：S852.65[农业科学—基础兽医学]