摘要
建立日本脑炎病毒一步法实时环介导逆转录等温快速扩增方法。根据日本脑炎病毒序列保守的非结构区基因NS3设计4条特异引物,用于扩增SA14-14-2株、SA103株和GD06株日本脑炎病毒获得成功,在病毒感染的单层细胞和猪体内都可以检出病毒。与常规SYBR GreenI适时荧光RT-PCR方法比较和Ct值分析,用不同稀释度病毒RNA,两者敏感性相似,适时RT-PCR在模板浓度低时结果更稳定。FIP和BIP纯度会影响反应效果,反应时间和模板浓度会影响电泳条带。一般63℃～65℃30min可出现结果,模板浓度低时要适当延长反应时间。以SYBR GreenI为指示剂,RT-LAMP在63℃～65℃循环扩增检测日本脑炎病毒,1.5min/循环,30个循环,Ct值为21.5。提示RT-LAMP方法用于日本脑炎诊断、鉴别,是敏感、可靠、成本低廉的方法,有助于人畜日本脑炎的防控工作。
A one step, accelerated reverse transcription loop-mediated isothermal amplification （RT-LAMP） procedure was developed for the detection of Japanese encephalitis virus（JEV）. The four primers required for RT-LAMP were designed using a conserved region in the NS3 of the nonstructural region of JEV. RT- LAMP was used to detect isolates of three strains of JEV including the strains JEV SA14-14-2, SA103 and GD06. The virus was detected reliably in both infected vero cell monolayer and swine hosts. RT-LAMP was compared to real-time RT-PCR with SYBR Green I and melting curve analysis, using serial dilutions of total RNA extracts. Similar sensitivities were observed, except that real-time RT-PCR was more con- sistent at lower template concentrations. The purity of the FIP and BIP primers affected the efficiency of the reaction, and incubation time and template concentration affected the ladder-like pattern observed after agarose gel electrophoresis. Ahhough JEV could be detected after 30 min of incubation at 63 ℃-65℃, a longer incubation time was required for lower concentrations of the target. The Ct value of real-time RT- LAMP（SYBR Green I）（30×1.5min ） was about 21.5. RT-LAMP is a very sensitive, low cost diagnostic tool that should be of value in more accurate determination of the distribution of JEV.
出处
《动物医学进展》
CSCD
北大核心
2009年第12期34-39,共6页
Progress In Veterinary Medicine
关键词
日本脑炎病毒
环介导逆转录等温扩增
快速检测
荧光定量
Japanese epidemic virus~ reverse transcription loop-mediated isothermal amplification （RT- LAMP）
rapid detection
real-time PCR
