应用重组酶介导逆转录扩增技术快速登革病毒血清型鉴定

Rapid serotype detection for Dengue virus by recombinase-aid reversetranscription amplification

目的本研究建立了基于逆转录重组酶介导核酸扩增技术(RT-RAA)快速鉴定登革病毒血清型的方法。方法选取登革病毒基因组NS1基因片段分别设计DV I型、DVⅡ型、DVⅢ型和DVⅣ型引物和探针,建立登革病毒血清型鉴定RT-RAA检测方法,并分别对其灵敏度、特异性和重复性进行测试。结果建立的方法检测时间短(<30min),与基孔肯雅病毒、黄热病毒、寨卡病毒、乙型脑炎病毒等黄病毒属病毒及DV不同血清型间无交叉反应,检测的灵敏度可达102copies/μl,试验的重复性好。结论建立的登革病毒血清型鉴定RT-RAA方法具有快速、特异以及灵敏的特点,为探究登革病毒是否本土化,确定登革病毒分子流行病学趋势提供技术支持。

ObjectⅣe To establish a rapid method for serotyping of Dengue virus by reverse transcriptase recombinase-aid amplification(RT-RAA).Methods The specific primers and probes for DV I、DVⅡ、DVⅢand DVⅣwere designed respectⅣely based on conserved genome sequences NS1,and the RT-RAA method for DV serotype detection was established.The sensitⅣity,specificity and stability of these primers and probes were evaluated differentiately.Results RT-RAA was performed for DV serotype detection with a short time(<30 minutes).The approach is also shown very specific as it had no cross-reaction with other related viruses,such as Chikungunya virus,Yellow fever virus,ZIKV virus,Japanese encephalitisvirus,and different serotypes of DV as well.Our results indicated that the amplification is very sensitⅣe which can detect 100 copies/μl.Conclusion This RT-RAA assay was rapid,sensitⅣe and specific.and might be used for exploring the localization of dengue virus and determining the molecular epidemiological trend of dengue virus.