摘要
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 In this study, we establish a one - step reverse transcriptase recombinase aid amplification（ RT - RAA） assay for yellow fever virus detection based on recombinase aid amplification （RAA）. Methods According to the conserved genome sequence design and probes of astrovirus, a RT - RAA was constructed, and the repetitiveness, specificity and sensitivity of it were evaluated. Yellow fever virus sample was detected by the constructed method and the results were verified with gene sequencing. Results The yellow fever virus was amplified with RT - RAA, and 40 U reverse transcriptase was added into system for amplification in the best. RT- RAA was performed with a short detection time（〈 20 min） and low detection limit（ 100 copy）. This method had no cross -reaction with dengue virus, West Nile virus, Japanese encephalitis virus and chikungunya virus, which showed good specificity. Conclusion This RT - RAA assay was rapid, specific and sensitive. It can be used for the rapid detection of yellow fever virus on port.
出处
《中国卫生检验杂志》
CAS
2016年第1期1-3,共3页
Chinese Journal of Health Laboratory Technology
基金
国家质检总局科技计划项目(2014IK062)
浙江出入境检验检疫局科技计划项目(ZK2014034)
关键词
重组酶介导扩增
黄热病毒
分子检测
Recombinase aid amplification
Yellow fever virus
Molecular detection
