犬圆环病毒实时荧光定量PCR检测方法的建立

发布时间：2018-03-28 22:26

  本文选题：犬圆环病毒　切入点：SYBR　出处：《动物医学进展》2017年05期

【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)。
[Abstract]:Dog circovirus (DogCVV) is a newly discovered circovirus in the family of circoviridae. The aim of this study was to establish a real-time fluorescent quantitative PCR assay for the detection of canine circovirus. The conserved region of ORF2 gene of DogCVV was cloned by PCR method. The standard positive plasmid pClon007-ORF2 was constructed. The fluorescent quantitative PCR reaction system was optimized by using standard positive plasmid pClon007-ORF2 as template. The results showed that the established method had a good linear relationship with the standard sample in the range of 4.67 脳 10 ~ (9) copies/ 渭 L ~ (4.67) 脳 10 ~ (-3) copies/ 渭 L. The correlation coefficient was 0.998, the slope was -3.4455.The sensitivity of this method was 4.67 脳 10~1copies/ 渭 L. there was no cross reaction in the detection of rabies virus, canine parvovirus, canine distemper virus, canine parainfluenza virus and canine adenovirus type 2. All the dilution standard sample templates showed narrow specific melting peak at 83.35 鈩，

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