猪细小病毒7型SYBR Green Ⅰ荧光定量PCR检测方法的建立及临床样品的检测

Diagnosis and detection of PPV7 by SYBR Green Ⅰ real-time PCR

采用PCR方法扩增猪细小病毒7型(PPV7)衣壳蛋白基因保守区域493 bp,将其克隆到pMD18-T载体,构建重组质粒作为标准阳性质粒,以其为模板建立用于检测PPV7的SYBR GreenⅠ实时PCR检测方法,并验证其灵敏性、特异性和重复性。结果表明,本试验建立的SYBR GreenⅠ荧光定量PCR检测方法在2.85×10^1~2.85×10^8拷贝/μL呈良好的线性关系,相关系数为0.995,斜率为4.158,灵敏度可达到2.85×10^1拷贝/μL。该方法对于猪繁殖障碍性传染病常见病原如PRRSV、PCV2和PRV未出现特异性扩增,表明特异性好。所有标准曲线在溶解温度为83.103℃时出现单一的特异峰。使用本方法检测了2017年山东地区7家养猪场216份临床样品,总阳性率为12.5%。

To establish a real-time fluorescence quantitative PCR method which can quickly and accurately detect PPV7.The conserved region of capsid protein gene was amplified by PCR and cloned into pMD18-T vector,which was used as template to optimize assay condition of developing a SYBR Green Ⅰ real-time PCR for detection of PPV7.The standard curve produced a good linear relationship between concentrations of the standard template at a range of 2.85×108-2.85×101 copies/μL,and the correlation coefficient and slope were 0.995 and 4.158,respectively.The sensitivity of this method was 2.85×101 copies/μL.The specificity assay showed no amplification of PRRSV,PCV2 and PRV.All standards were specific narrow melting peak at 83.103℃.The positive rates were 12.5% in detected samples in Shandong in 2017.The results showed that SYBR Green Ⅰ PCR was a rapidly specific and sensitive method for the detection of PPV7.