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目的本研究采用重组酶介导的等温核酸扩增方法(RAA),通过使用逆转录酶,建立黄热病毒的一步法等温核酸扩增(RT-RAA)方法。方法根据黄热病毒基因组保守序列设计引物和探针,建立并分析RT-RAA的重复性、特异性、灵敏度;以所建立方法对黄热病毒样本进行检测,同时以基因测序进行验证。结果黄热病毒RT-RAA扩增,体系中加入40 U的逆转录酶扩增效果最佳。该方法检测时间短(<20 min),并且灵敏度高,检测下限可达100 copy,与登革病毒、西尼罗病毒、日本乙型脑炎病毒、基孔肯雅热病毒等蚊媒病毒无交叉反应,具有良好的特异性。结论构建的黄热病毒RT-RAA方法具有快速、特异以及灵敏的特点,适应于黄热病毒的口岸快速检测。
Objective To establish a one-step isothermal nucleic acid amplification of yellow fever virus (RT-RAA) by reverse transcriptase using a recombinant enzyme-mediated isothermal nucleic acid amplification method (RAA). Methods The primers and probes were designed according to the conserved sequences of the yellow fever virus genome to establish and analyze the repeatability, specificity and sensitivity of RT-RAA. The yellow fever virus was detected by the established method and verified by gene sequencing. Results Yellow fever virus RT-RAA amplification, the system by adding 40 U reverse transcriptase amplification best. The method has the advantages of short detection time (<20 min), high sensitivity and a lower detection limit of up to 100 copy. The method has the same advantages as dengue virus, West Nile virus, Japanese JE virus and Chikungunya fever virus Cross-reaction, with good specificity. Conclusion The constructed yellow fever virus RT-RAA method is rapid, specific and sensitive, and is suitable for rapid detection of yellow fever virus at the port.