SYBR GreenⅠ实时定量PCR方法检测猪细小病毒

Establishment of SYBR Green Ⅰ real-time PCR assay for the detection of porcine parvovirus

为建立特异敏感的猪细小病毒(PPV)的SYBR GreenⅠ实时定量PCR检测方法,本研究根据GenBank上PPV NS1保守序列设计合成了1对引物,利用本实验室构建的重组质粒(pMD18-T-PPV NS1)为标准模板,对反应条件进行优化,绘制标准曲线,并进行熔解曲线分析,建立了PPV的SYBR GreenⅠ实时定量PCR检测方法。分别进行了特异性、敏感性及重复性检验,结果表明,本方法具有良好的特异性,与PRRSV、CSFV、PRV、PCV-2、SIV及阴性对照均为阴性;其最低检出量为7copies/μL,比PCR敏感100倍,且批内和批间重复性良好。用该方法对15份临床病料进行检测,并与常规PCR、环介导等温扩增(LAMP)方法进行对比,显示该方法灵敏度高、成本低,能够对样品组织中病毒进行定量检测,为快速检测PPV提供了有效的技术手段。

A SYBR GreenⅠreal-time PCR for detecting porcine parvovirus（PPV） was established using primers derived from the published sequences.A plasmid containing pMD18-T-PPV NS1 was constructed and served as a standard template to generate the standard curve.The assay had high specificity and could detect as low as 7 copies of virus DNA.The sensitivity was 100 times greater than that of general PCR.Fifteen clinical samples were detected by this assay,conventional PCR and LAMP at the same time. The results showed that PPV SYBR Green Ⅰ real-time PCR assay is highly sensitive and of low cost.It could be used as an effective tool for the rapid detection of clinical samples.