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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)。
Canine Circovirus (DogCV) is a newly discovered circovirus of the family of circovirus that aims to establish a real-time quantitative PCR assay for canine ring virus. In this study, we cloned the conserved region of ORF2 of canine circovirus by PCR and constructed the standard plasmid pClon007-ORF2. The standard positive plasmid pClon007-ORF2 was used as a template to optimize the fluorescence quantitative PCR reaction system. The results showed that the established method showed a good linear relationship with the standard of 4.67 × 10 ~ 9 copies / μL ~ 4.67 × 10 ~ 3 copies / μL with a correlation coefficient of 0.998 and a slope of -3.445. The detection sensitivity of this method is 4.67 × 10 ~ 1copies / μL. The method for rabies virus, canine parvovirus, canine distemper virus, canine parainfluenza virus, canine adenovirus type 2 canine common pathogen detection no cross-reaction; all dilution standard template at 83.35 ℃ appear narrow specificity Sexual melting peak. The detection of clinical samples showed that the positive rate of canine circovirus detected by real-time PCR was 12.50% (4/32).