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


CONCLUSIONS
This TaqMan-based one-step RT-PCR assay was a quick, sensitive and specific tool for molecular diagnosis of Japanese encephalitis virus.
Xie
Rong-Hui
RH
Key Lab of Vaccine Against Hemorrhagic Fever with Renal Syndrome, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China.
Xu
Fang
F
Zhu
Han-Ping
HP
Cheng
Yin-Kai
YK
Fu
Gui-Ming
GM
Yao
Ping-Ping
PP
Weng
Jing-Qing
JQ
Lu
Yi-Yu
YY
Yang
Zhang-Nv
ZN
Zhu
Zhi-Yong
ZY
D000818:Animals; D009033:Culicidae / Q000821:virology*; D004664:Encephalitis Virus, Japanese / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012367:RNA, Viral