Evaluation of the accuracy of one step Real-time RT-PCR on detecting West Nile virus

Objective The accuracy including sensitivity and specificity of the one step real-time reverse transcriptase PCR that have already been established for detecting West Nile virus was systemically evaluated with different genera of Flavivirus virus in the present study,rendering it more reliable and accurate in application of this method to detect West Nile virus not only in laboratory diagnosis but also epidemiological investigation. Methods The probe for the one step real-time reverse transcriptase PCR was designed from the alignment of the sequences of West Nile virus lineage 1 strains and lineage 2 strains using Vector NTI Suite software Ver. 8.0. The sensitivity of this method was evaluated by the Vero cell and BALB/c mouse brain tissue infected with West Nile virus,while the specificity was determined with other different Flavivirus sp. of the family Flaviviridae,such as Japanese encephalitis virus,Dengue virus,tick borne encephalitis virus,and also eastern equine encephalomyelitis virus belonging to the genus Alphavirus of the family Togaviridae. Results The one step real-time RT-PCR method could be utilized for detecting most of West Nile virus lineage 1 strains with high sensitivity,and the lowest detectable concentration of West Nile virus was calculated as 5.75×10-2pfu/ml. No cross reactions were observed with Japanese encephalitis virus,Dengue virus,tick borne encephalitis virus and eastern equine encephalomyelitis virus. Conclusion The method is characterized with high sensitivity and specificity,and is accurate enough for the application of quantitative detection of West Nile Virus RNA for epidemiological survey and laboratory studies.