Nest RT-PCR to detect the West Nile virus

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.