西尼罗病毒套式RT-PCR检测方法的建立

Nest RT-PCR to detect the West Nile virus

从GenBank上调取西尼罗病毒的基因序列,经过分析,设计并合成2套套式RT-PCR引物,分别对西尼罗病毒灭活苗进行扩增,结果扩增出与目的片段大小一致的条带,将其克隆入pMD18-T-Vector中,进行序列测定,证实为WNV的基因。建立了套式RT-PCR检测方法,经各反应条件的优化后,发现2套nest-PCR引物能分别扩增出85个拷贝和62个拷贝的双链DNA,对乙型脑炎病毒、黄热病毒等11种病毒核酸进行扩增,发现具有良好的特异性,显示建立套式RT-PCR具有高效、快速、特异、灵敏的特点,可用于口岸WNV的检测和监测。

Two pairs of nest-PCR primers were designed and synthesized based on the nucleotide sequences of the West Nile virus in GenBank. After optimization of the detection methods, they were used to amplify the West Nile virus inactivated vaccine. The result showed that all primers got the specific bands. Then the two fragments from the RT-PCR products were cloned into pMD18-T-vector, respectively, sequenced and confirmed to be West Nile virus gene. The maximal detection limits by two pairs of nest-PCR primers were approximately 85 copies and 62 copies of two strands DNA, respectively. The high specificity of the method was demonstrated by detecting 11 viruses, such as Japanese encephalitis virus, Yellow Fever virus, and so on. Therefore, two nest-PCR methods developed here for WNV detection had the characteristic of high efficiency, speediness, specificity and sensitivity. Thereby it can be used to entry-exit inspection.