[Development and evaluation of TaqMan-based one-step reverse transcription-polymerase chain reaction assay for the detection of Japanese encephalitis virus].

ABSTRACT

OBJECTIVE: To establish a TaqMan based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of Japanese encephalitis virus. METHODS: The gene sequences of Japanese encephalitis virus downloaded from the GenBank was aligned, using the biologic software. Specific primers and probes were designed in the conserved region of the C gene for Japanese encephalitis virus. The real-time RT-PCR reactive condition was optimized and the sensitivity, specificity and the stability of the assay were evaluated. Mosquitoes collected from Zhejiang province were detected by this assay. RESULTS: Mg2+, primer and probe were optimized at 5 mmol/L, 0.2 micromol/L and 0.1 micromol/L respectively. The specificity of the assay was high and there were no cross reactions with dengue virus, rabies virus, seoul virus or hantan virus. The detection limits of the assay was 0.1 TCID50. Results from preliminary application showed that TaqMan RT-PCR for Japanese encephalitis virus was sensitive, easier and faster to perform the process of traditional virus isolation and identification. It took only three hours to extract viral RNA and perform the real-time RT-PCR. CONCLUSION: This TaqMan-based one-step RT-PCR assay was a quick, sensitive and specific tool for molecular diagnosis of Japanese encephalitis virus.