新疆出血热病毒的实时荧光RT-PCR检测方法研究

Research on Real time RT-PCR for Xinjiang hemorrhagic fever virus

目的:建立新疆出血热病毒的实时荧光RT-PCR检测方法。方法:分析新疆出血热病毒核酸S片段序列的保守性,设计新疆出血热病毒实时荧光RT-PCR引物和探针。以体外转录后的RNA作为阳性对照模板,摸索出实时荧光RT-PCR检测的最佳引物浓度、探针浓度和反应体系条件,并进行灵敏度和特异性分析。结果:建立的新疆出血热病毒的实时荧光RT-PCR检测方法最佳反应体系为:正向引物CCHFV-FP终浓度250 nM,反向引物CCHFV-RP终浓度500 nM,探针CCHFV-Probe终浓度500 nM;扩增程序为:50℃10 min,95℃10 min;95℃5 s,57℃20 s 45次循环。该方法最低检测限约为2 copies病毒/反应,与森林脑炎病毒和汉坦病毒无交叉反应。结论:该方法灵敏度高、特异性强,适用于新疆出血热病毒的快速检测。

Objective：To set up real time RT-PCR method for Xinjiang hemorrhagic fever virus.Methods： S segment sequences of Xinjiang hemorrhagic fever virus nucleic acid were analyzed.Primers and probes were designed in conserved region and used for real-time RT-PCR.With in vitro transcription of RNA as a positive control template,the appropriate primer concentration,probe concentration and reaction conditions were determined.The sensitivity and specificity of real-time PCR detection were also done.Results： The optimized reaction system involved 250 nM CCHFV-FP,500 nM CCHFV-RP and 500 nM CCHFV-Probe.Real time RT-PCR amplification programs were reverse transcription at 50℃ for 10 min and denaturation at 95℃ for 10 min followed by 45 cycles at 95℃ 5 s and 57℃ 20 s.The detection limit was 2 copies virus per reaction.It had no cross-reaction with forest encephalitis virus and hantavirus.Conclusion： The established real time RT-PCR method has high sensitivity and strong specificity,which is suitable for rapid detection of Xinjiang hemorrhagic fever virus.