犬瘟热病毒、犬细小病毒二重TaqMan MGB探针实时荧光定量PCR检测方法的建立与应用  

Establishment and Application of A Double TaqMan MGB Real-time PCR Assay for Detection of Canine Distemper Virus and Canine Parvovirus

作　　者：赵雪丽[1] 刘毅[2] 班付国[1] 闫若潜[1] 王华俊[1] 王淑娟[1] 马震原[1] 王东方[1] ZHAO Xueli1 , LIU Yi2 , BAN Fuguo1 , YAN Ruoqian1 , WANG Huajun1 , WANG Shujuan1 , MA Zhenyuan1 , WANG Dongfang1(1. Henan Centre for Animal Disease Control ＆Prevention, Zhengzhou 450008, China; 2. China Animal Disease Control Centre, Beijing 100125, Chin)

机构地区：[1]河南省动物疫病预防控制中心,郑州450008 [2]中国动物疫病预防控制中心,北京100125

出　　处：《中国畜牧兽医》2018年第8期2086-2094,共9页China Animal Husbandry & Veterinary Medicine

基　　金：河南省科技创新人才计划项目(174200510003)

摘　　要：为建立一种特异、高通量检测犬瘟热病毒(canine distemper virus,CDV)和犬细小病毒(canine parvovirus,CPV)的二重TaqMan MGB探针实时荧光定量PCR方法,本研究选取高度保守且具有型特异性的CDV H基因和CPVVP2基因序列,设计CDV和CPV特异性引物对及TaqMan MGB探针;经反应条件优化,建立了检测CDV和CPV的二重TaqMan MGB探针实时荧光定量PCR方法,并进行了敏感性、特异性和重复性试验。结果显示,该方法检测CDV、CPV的标准曲线线性相关系数(R^2)分别为0.997和0.993,最低检出限均为10拷贝/μL,具有较高的灵敏性;对犬副流感病毒等4种病原对照和阴性对照均未出现扩增,特异性良好;CDV、CPV标准品批间试验结果显示,该方法具有很好的重复性;对48份临床疑似CDV或CPV感染样品检测结果显示,14份为CDV阳性,19份为CPV阳性,4份为CDV/CPV双阳性,与CDV H基因、CPVVP2基因测序结果符合率为100%。本研究建立的二重TaqMan MGB探针实时荧光定量PCR方法具有灵敏、特异、高通量和可准确定量等优点,可用于临床CDV/CPV病毒感染各个时期的快速鉴别检测。This study was aimed to establish a double TaqMan MGB Real-time PCR assay to simultaneously and specifically detect canine distemper virus（CDV）and canine parvovirus（CPV）in one reaction.Two pairs of specific primers for CDV and CPV,along with two TaqMan MGB probes for each virus were designed in the assay basing on CDV H gene and CPVVP2 gene sequences.The specificity,sensitivity and repetition of the double TaqMan MGB Real-time PCR assay were tested,and 48 samples taken from clinic suspicious CDV and CPV infected canines had been testified by the established double TaqMan MGB Real-time PCR.The results indicated that the doulde TaqMan MGB Real-time PCR assay was successfully established,and the number of standard curve correlation（R^2）of CDV and CPV were 0.997 and 0.993,respectively.The speci-ficity of the double TaqMan MGB Real-time PCR assay revealed that amplifications were showed on CDV and CPV samples,but other pathogens and negative controls had no amplifications;The sensitivity of CDV and CPV were both 10 copies/μL.Meanwhile,14 CDV positive samples,19 CPV positive samples and 4 CDV/CPV double positive samples were detected,which were consistent with the results of the sequencing.Therefore,the established double TaqMan MGB Realtime PCR assay had high sensitivity,specificity and flux accurate quantitative,which could be applied to clinical CDV/CPV infection each periods.

关 键 词：犬瘟热病毒(CDV) H基因 犬细小病毒(CPV) VP2基因 二重TaqMan MGB探针实时荧光定量PCR 

分 类 号：S852.655[农业科学—基础兽医学]