犬细小病毒Taq Man荧光定量PCR检测方法的建立与初步应用  

Establishment and preliminary application of a Taq Man-based real-time PCR method for detection of canine parvovirus

作　　者：刘海防[1] 胡传伟[2] 谢之景[1] 姜世金[1] 张兴晓[1] 倪长鹏[2] 贾赟[2] 杨笃宝[1] 

机构地区：[1]山东农业大学动物科学院,山东泰安271018 [2]辽宁出入境检验检疫局技术中心,辽宁大连116001

出　　处：《中国兽医学报》2010年第5期593-596,共4页Chinese Journal of Veterinary Science

基　　金：山东农业大学青年科技创新基金资助项目(23204);国家质检总局基金资助项目(2005IK047)

摘　　要：根据GenBank中CPV VP2蛋白基因序列,选择CPV VP2基因保守序列,分别设计1对特异性引物和TaqMan探针,建立了CPV Taq Man荧光定量PCR检测方法。对CPV Taq Man荧光定量PCR的反应体系和反应条件进行优化,并对其特异性和敏感性进行了测定,建立标准曲线。结果,CPV Taq Man荧光定量PCR对CPV检测阳性,并能对10拷贝/μL的CPV模板检测阳性,标准曲线的相关系数为0.9992,但对非CPV模板检测阴性。结果表明,所建立的CPV Taq Man荧光定量PCR具有很好的特异性和敏感性,标准曲线具有很强的实用性。同时对CPV Taq Man荧光定量PCR与CPV PCR进行了比较试验,结果前者比后者的敏感性高100倍。采用CPVHA、CPV PCR与CPV Taq Man荧光定量PCR对55份临床样品进行检测,CPV HA对30份样品检测阳性,CPVPCR对40份样品检测阳性,CPV Taq Man荧光定量PCR对47份样品检测阳性。这表明,CPV Taq Man荧光定量PCR对样品的检出率最高,具有特异、敏感、快速的特点,适用临床样品的检测。To establish a Taq Man-based real-time PCR method for detection of canine parvovirus,the pair of primers and the Taq Man probe were designed according to the VP2 genes of canine parvovirus in Genbank.The reactive conditions of the real-time PCR method were optimized to improve its specificity and sensitivity,and the correlation coefficient of standard curve was 0.999 2.As a result,the specificity and sensitivity of the real-time PCR method were very good and the CPV sample including 10 copies/μL was positively tested with it,the no-CPV samples negetivly.The standard curve was practical.The sensitivity of the method was comparatively higher than that of CPV PCR by 100×.With CPV HA,CPV PCR and the real-time PCR method,the 55 clinical samples were tested,and 47 samples were checked up on with the real-time PCR method,40 samples positive with CPV PCR,30 samples positive with CPV HA.It implied that the real-time PCR method was specific and sensitive,simplified matters,and may be used in clinical diagnosis.

关 键 词：犬细小病毒 实时定量PCR TAQ Man荧光探针 

分 类 号：S852.65[农业科学—基础兽医学]