犬圆环病毒实时荧光定量PCR检测方法的建立  

Development of a Real-time PCR Method for Detection of Dog Circovirus

作　　者：孙文超[1,3] 解长占 赵冠宇[3] 曹亮[3] 郑敏[2] 曹慧慧[1,2] 张红云 韦显凯[2] 韩继成[3] 温树波[3] 肖朋朋[3] 靖杰[3] 张萍[3] 鲁会军[3] 金宁一[3] 

机构地区：[1]广西大学动物科技学院,广西南宁53001 [2]广西动物疫病预防控制中心,广西南宁530001 [3]军事医学科学院军事兽医研究所,吉林长春130122 [4]广西玉林市动物疫病预防控制中心,广西玉林537000

出　　处：《动物医学进展》2017年第5期6-10,共5页Progress In Veterinary Medicine

基　　金：广西自然科学基金项目(2012GXNSFAA053073)

摘　　要：犬圆环病毒(DogCV)是圆环病毒科新发现的圆环病毒,旨在建立犬圆环病毒实时荧光定量PCR检测方法。试验用PCR方法克隆犬圆环病毒ORF2基因保守区域,构建标准阳性质粒pClon007-ORF2。以标准阳性质粒pClon007-ORF2为模板对荧光定量PCR反应体系进行优化。结果表明,所建立的方法 Ct值与标准品在4.67×10~9 copies/μL^4.67×10~3 copies/μL范围内呈良好的线性关系,相关系数为0.998,斜率为-3.445。该方法检测灵敏度为4.67×10~1copies/μL。该方法对狂犬病病毒、犬细小病毒、犬瘟热病毒、犬副流感病毒、犬腺病毒2型等犬常见病原的检测均无交叉反应;所有稀释度标准品模板均在83.35℃出现窄的特异性熔解峰。临床样品检测表明,所建立的实时荧光定量PCR对犬圆环病毒的阳性检测率为12.50%(4/32)。Dog circovirus was found as a member of the family Circoviridae.This study was to establish a real-time fluorescence quantitative PCR method which can detect dog circovirus quickly and accurately in clinic.The conserved region of the ORF2 gene of dog circovirus was amplified by PCR and cloned into pClon007 vector.The pClon007-ORF2 plasmid DNA was used as template to optimize assay condition of developing a SYBR Green I real-time PCR for detection of dog circovirus.The standard curve produced a good linear relationship between Ct value and initial amounts of total DNA at a range of 4.67 × 109-4.67 × 103 copies per microlitre,and the correlation coefficient and slope were 0.998 and --3.445 respectively.The sensitivity of this method was 4.67×101 copies/μL. The specificity assay showed no amplification of RABV, CPV, CDV, CPIV, CAV-2. All standards are specific narrow melting peak at 83.35 ℃. The positive rates were 12.50%（4/32） of samples from Guangxi.

关 键 词：犬圆环病毒 SYBR Green Ⅰ 实时荧光定量PCR 

分 类 号：S852.659.2[农业科学—基础兽医学]