西尼罗病毒荧光定量PCR检测体系的建立及评价

本研究建立了一种实时荧光定量PCR快速核酸检测西尼罗病毒的方法。通过序列比对和blast分析，确定西尼罗病毒Caspid蛋白保守区基因为检测的目的基因，引物采用Primer Premier5.0软件进行设计。本研究建立的检测方法利用SYBR Green染料，相比探针成本较低。通过溶解曲线分析表明，建立的检测方法在扩增过程中没有发现有二聚体的产生。本检测体系在用空白对照及类似的乙脑病毒作为扩增对照时，没有发现非特异性产物的生成，表明该体系对于西尼罗病毒的检测是特异的。将阳性对照标准品进行10倍梯度稀释后可检测到102copies/μL样品，表明该检测体系具有较高的检测灵敏度。

Establishment and Evaluation of Real-time PCR for West nile virus Detection

A rapid real-time polymerase chain reaction (RT-PCR) for detection of WNV was established in this study. Primers were designed according to capsid protein gene with Primer Premier5.0. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green. Amplifying curve showed that this method could successfully amplify WNV gene, nevertheless reference Japanese encephalitis and blank control were all negative. 10-fold dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The newly-built real-time PCR has high sensitivity, good specificity, reliable stability, so it has potential to apply in inspection and quarantine of WNV.