猪圆环病毒2型SYBR GreenⅠ荧光定量PCR诊断方法的建立

Establishing a SYBR GreenⅠ Real-time Quantitative PCR Assay for Detecting PCV-2

根据Gen Bank中猪圆环病毒2型ORF2基因序列设计特异性引物,PCR扩增获得猪圆环病毒2型ORF2基因片段,并克隆到p MD-18T载体上作为阳性标准品。通过对SYBR Green I荧光定量PCR反应条件的优化,建立了猪圆环病毒2型的SYBR Green I荧光定量PCR诊断方法。扩增产物的溶解曲线分析只出现1个单特异峰,无引物二聚体,对PRV、PPV、E.coli、CSFV、PRRSV均无阳性信号,可重复性好,测灵敏度可达4.0×101拷贝/μL。结果表明,建立的猪圆环病毒2型SYBR Green I实时荧光定量PCR具有特异、灵敏、快速、重复性好,适合于猪圆环病毒2型临床样品的检测。

Based on the ORF2 gene sequences of PCV-2 in Gen Bank, one pairs of specific primer was designed to amplify the specific fragments of ORF2 gene and then the amplified ORF2 gene of PCV-2 was cloned into p MD-18 T vector to prepare positive standard. After optimizing annealing temperature and primers concentrations, a SYBR Green I Real-time Quantitative PCR was established for detecting PCV-2. The melting curve analysis using SYBR Green I dye showed one specific peak. There was no primer-dimers peak, no amplification from PRV,PPV, E.coli, CSFV and PRRSV. The PCR was highly sensitive to 4.0 ×10^1copies/μL DNA. It is indicated that the SYBR Green I Real-time Quantitative PCR assay established was sensitive, specific, rapid and highly repeatable. It can be used to detect PCV-2 in clinical samples.