摘要
根据GenBank中猪细小病毒VP2基因序列设计特异性的引物,PCR扩增获得猪细小病毒VP2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,建立了猪细小病毒的SYBR Green I荧光定量PCR诊断方法,以此为基础研制出试剂盒。试剂盒扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,对PRV、PCV-2、E.coli、CSFV、PRRSV均无阳性信号扩增,可重复性好,测灵敏度可达1.0×101拷贝/μL。结果表明,研制的猪细小病毒SYBR Green I实时荧光定量PCR试剂盒具有特异、灵敏、快速、重复性好等优点,适合于猪细小病毒临床样品的检测。
Based on the VP2 gene sequences of PPV in GenBank, one pairs of specific primer was designed for amplifying the specific fragments of VP2 gene. The amplified VP2 gene of PPV was cloned into p MD-18 T vector and used as positive standard. After optimizing annealing temperature and primers concentrations, a SYBR Green I fluorescent Quantitative PCR was established for simultaneously detecting PPV. The melting curve analysis using SYBR Green I dye showed one specific peak. No primer-dimers peak was observed. No amplification was amplified from PRV, PCV-2, E.coli, CSFV, PRRSV with the SYBR Green I Real-time Quantitative PCR. The PCR kit was highly sensitive to 1.0×101copies/μL DNA. It is revealed that the SYBR Green I Real-time Quantitative PCR kit was sensitive, specific with good repetition. It could be used to detect PPV in clinical samples.
出处
《湖北农业科学》
2015年第1期126-129,133,共5页
Hubei Agricultural Sciences
基金
贵州省农业攻关项目[黔科合NY字(2010)3085]
贵州省畜禽健康养殖技术创新能力建设项目[黔科合院所创能(2010)4004]
