Development and evaluation of TaqMan-based one-step reverse transcription-polymerase chain reaction assay for the detection of Japanese encephalitis virus
10.3760/cma.j.issn.0254-6450.2009.03.019
- VernacularTitle:一步法流行性乙型脑炎病毒TaqMan荧光定量反转录聚合酶链反应方法的建立及应用
- Author:
Rong-Hui XIE
1
;
Fang XU
;
Han-Ping ZHU
;
Yin-Kai CHENG
;
Gui-Ming FU
;
Ping-Ping YAO
;
Jing-Qing WENG
;
Yi-Yu LU
;
Zhang-Nv YANG
;
Zhi-Yong ZHU
Author Information
1. 浙江省疾病预防控制中心
- Keywords:
Japanese encephalitis virus;
Real-time reverse transcription-polymerase chain reaction;
Detection
- From:Chinese Journal of Epidemiology
2009;30(3):277-280
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a TaqMan based real-time reverse transeription-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 μmol/L and 0.1 μmol/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.
