Xie Rong-Hui, Xu Fang, Zhu Han-Ping, Cheng Yin-Kai, Fu Gui-Ming, Yao Ping-Ping, Weng Jing-Qing, Lu Yi-Yu, Yang Zhang-Nv, Zhu Zhi-Yong
Key Lab of Vaccine Against Hemorrhagic Fever with Renal Syndrome, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China.
Zhonghua Liu Xing Bing Xue Za Zhi. 2009 Mar;30(3):277-80.
To establish a TaqMan based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of Japanese encephalitis virus.
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.
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.
This TaqMan-based one-step RT-PCR assay was a quick, sensitive and specific tool for molecular diagnosis of Japanese encephalitis virus.
建立一种基于TaqMan的实时逆转录-聚合酶链反应（RT-PCR）检测方法用于检测日本脑炎病毒。
从GenBank下载日本脑炎病毒的基因序列，使用生物软件进行比对。在日本脑炎病毒C基因的保守区域设计特异性引物和探针。优化实时RT-PCR反应条件，并评估该检测方法的灵敏度、特异性和稳定性。用此检测方法对从浙江省采集的蚊子进行检测。
Mg2+、引物和探针分别优化至5 mmol/L、0.2 μmol/L和0.1 μmol/L。该检测方法特异性高，与登革病毒、狂犬病病毒、汉城病毒或汉坦病毒无交叉反应。该检测方法的检测限为0.1 TCID50。初步应用结果表明，用于检测日本脑炎病毒的TaqMan RT-PCR比传统的病毒分离和鉴定方法更灵敏、操作更简便快捷。提取病毒RNA并进行实时RT-PCR仅需3小时。
这种基于TaqMan的一步法RT-PCR检测方法是用于日本脑炎病毒分子诊断的快速、灵敏且特异的工具。
