猪肠道α冠状病毒荧光定量PCR检测方法的建立

Development of a SYBR Green I real-time PCR method for detection of SeACoV

为建立猪肠道α冠状病毒(SeACoV)荧光定量检测方法,本研究采用RT-PCR方法扩增SeACoV N基因保守区序列,将其克隆至p EASY-Blunt载体,构建重组质粒p EASY-Blunt-PA-N作为标准阳性质粒,以其为模板建立SeACoV的SYBR Green I荧光定量PCR检测方法,并进行特异性、敏感性和重复性试验验证。结果显示,建立的SYBR Green I荧光定量PCR检测方法 Ct值与标准品模板在9.47×101拷贝/μL～9.47×107拷贝/μL范围内呈良好的线性关系,相关系数为0.996,斜率为-3.91。本实验建立的方法对PEDV、TGEV、PDCo V、PRRSV等猪常见腹泻病样品检测无扩增,表明特异性良好;敏感性试验结果显示,该方法检测下限可达到9.47×101拷贝/μL;重复性试验结果显示,组内变异系数在0.82%～1.01%,组间变异系数在1.20%～1.69%,重复性较好。采用本研究建立的方法对广西和云南等地70份临床样品进行检测,结果显示除阳性对照外,临床样品均为阴性,表明该病毒尚未传播至广东以外的省份。本实验首次建立了SeACoV N基因SYBR Green I荧光定量PCR检测方法,为SeACoV快速检测和病毒感染预防提供技术手段。

This study was to establish a real-time fluorescence quantitative PCR method which can quickly and accuratelydetect SeACoV. The conserved region of SeACoV N gene was amplified by PCR and cloned into pEASY-Blunt vector. Theresulted pEASY-Blunt-N gene plasmid DNA was used as template to optimize assay condition of developing a SYBT Green Ireal-time PCR for detection of SeACoV. The standard curve produced a linear relationship between Ct value and initial amounts oftotal DNA at a range of 9.47×101-9.47×107 copies/μL, the correlation coefficient and slope were 0.996 and-3.91, respectively.The specificity assay showed no amplification of PEDV, TGEV, PDCoV and PRRSV which displayed a good specificity;Thesensitivity of this method was 9.47×101 copies/μL;Repeatability analysis showed that the coefficient of variation within the groupranged from 0.82% to 1.01%;The coefficient of variation between groups ranged from 1.20% to 1.69%. A total of 70 clinical samples from Guangxi and Yunnan were tested by this method and no positive samples were detected. The results showed thatSYBR Green I real-time PCR was a rapidly specific and sensitive method for the detection of SeACoV.