鉴别犬瘟热病毒野毒株与疫苗株双重一步法RT-PCR检测方法的建立与应用  

Establishment and application of double one-step RT-PCR method for the differential detection of Canine distemper virus wild strains and vaccine strains

作　　者：梅胜尧 张玉堂 张逸民 孙宝海 刘晶 MEI Shengyao;ZHANG Yutang;ZHANG Yimin;SUN Baohai;LIU Jing(Jiangsu Drug Safety Evaluation Center,Jiangsu Drug Research Institute Co.,Ltd.,Nanjing 210018,China)

机构地区：[1]江苏省药物研究所有限公司江苏省药物安全性评价中心,南京210018

出　　处：《黑龙江畜牧兽医》2022年第8期75-81,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：江苏省实验动物协会2019年实验动物研究课题(DWXH201913)。

摘　　要：为了建立一种简单、快速鉴别检测犬瘟热病毒(CDV)野毒株与疫苗株的方法,试验根据Gen-Bank中CDV野毒株与疫苗株H基因序列设计鉴别检测引物,建立鉴别CDV野毒株与疫苗株一步法RT-PCR,并验证该方法的特异性、敏感性、重复性及符合性;利用建立的鉴别CDV野毒株与疫苗株双重一步法RT-PCR检测方法对2017—2020年采集于江苏省11个不同地市的505份发病犬喉拭子样品和病料样品进行检测,确定CDV野毒株与疫苗株双重一步法RT-PCR的临床适用性。结果表明:该方法对CDV野毒株、CDV疫苗株、CDV野毒株/疫苗株可分别扩增出477 bp、677 bp、477 bp/677 bp的特异性条带,而检测犬细小病毒(CPV)、犬腺病毒(CAV)、犬副流感病毒(CPIV)、犬冠状病毒(CCV)均为阴性;该方法的检测灵敏度为23.1 pg RNA;与RFLP方法的符合率为100%;批内重复性检测和批间重复性检测的结果完全一致;利用该方法检测505份临床样品,CDV野毒株和疫苗株的阳性率分别为8.91%和72.48%。说明建立的鉴别CDV野毒株与疫苗株一步法RT-PCR检测方法具有良好的特异性、敏感性、重复性、准确性和临床适用性。In order to establish a simple and rapid method for the differential detection of Canine distemper virus(CDV) wild strains and vaccine strains, in this experiment the differential detection primers were designed based on the H gene sequences of CDV wild strains and vaccine strains in GenBank, and one-step RT-PCR was established for CDV wild strains and vaccine strains, which verified the specificity, sensitivity, repeatability and compliance of the method. The established double one-step RT-PCR method of CDV wild strains and vaccine strains was used 505 throat swab samples and disease material samples of infected dogs collected from 11 different cities in Jiangsu Province from 2017 to 2020 were detected, to determine the clinical applicability of double one-step RT-PCR of CDV wild strains and vaccine strains. The results showed that the method could amplify the specific bands of 477 bp, 677 bp, 477 bp/677 bp for CDV wild strains, CDV vaccine strains, CDV wild/vaccine strain, respectively, while the detection results of Canine parvovirus(CPV), Canine adenovirus(CAV), Canine parainfluenza virus(CPIV), and Canine coronavirus(CCV) were all negative. The detection sensitivity of double one-step RT-PCR was 23.1 pg RNA;the coincidence rate of double one-step RT-PCR and RFLP method was 100%;the results of double one-step RT-PCR intra-assay and inter-assay repeatability were completely consistent. Using this method to detect 505 clinical samples, the positive rates of CDV wild strains and vaccine strains were 8.91% and 72.48%, respectively. The results suggested that the established one-step RT-PCR of CDV wild strains and vaccine strains had good specificity, sensitivity, repeatability, accuracy and clinical applicability.

关 键 词：犬瘟热病毒 野毒株 疫苗株 一步法RT-PCR 检测方法 应用 

分 类 号：S852.655[农业科学—基础兽医学] Q78[农业科学—兽医学]