摘要
目的:将重组酶介导等温扩增技术(RAA)和实时荧光探针相结合拟开发一种简易、快速的人腺病毒(HAdV)-11/55检测方法。方法:以HAdV-11/55的Hexon基因作为靶序列设计合成多对引物和探针,选择最佳引物探针组合,在39℃、40℃、42℃条件下进行重组酶介导等温扩增,选出最佳反应温度,并评估该方法的灵敏度和特异性。结果:实验表明该方法的最适温度为42℃,最低检测限为101 copies/μL。特异性检测结果显示,该实时荧光RAA检测方法可以检测HAdV-11/55,且不与其他亚型和其他呼吸道病原体发生交叉反应,特异性良好,该方法所得结果与实时荧光PCR检测结果一致性为100%,具有良好的检测性能。结论:本研究开发的实时荧光RAA检测方法可以在仅有简单恒温设备的条件下20 min内完成检测,是一种检测HAdV-11/55快速且简易的方法,适用于HAdV-11/55的床旁分子诊断检测。
Objective:To develop a simple and rapid detection method for HAdV-11/55 by combining recombinase aided amplification technology with real-time fluorescent probe.Methods:Using the Hexon gene of HAdV-11/55 as the target sequence,multiple pairs of primers and probes were designed and synthesized,and the best combination of primer and probe was selected.Recombinase-mediated isothermal amplification was carried out at 39℃,40℃and 42℃to select the best reaction temperature,and the sensitivity and specificity of the method were evaluated.Results:The optimal temperature of the method was 42℃.The minimum detection limit is 101 copies/μL.The specificity test results showed that the real-time fluorescent RAA detection method could detect HAdV-11/55,and there was no cross-reaction with other subtypes and other respiratory pathogens.The results of this method were 100%consistent with the results of real-time fluorescent PCR,which had excellent detection performance.Conclusions:The real-time fluorescent RAA assay developed in this study can be completed within 20 minutes with only simple thermostatic equipment.It is a rapid and simple method for detecting HAdV-11/55,which is suitable for point-of-care molecular diagnostic testing of HAdV-11/55.
作者
周华君
吴晓虹
赵治友
车飞虎
孙青阳
成军
戴玉柱
ZHOU Huajun;WU Xiaohong;ZHAO Zhiyou;CHE Feihu;SUN Qingyang;CHENG Jun;DAI Yuzhu(Department of Clinical LaboratoryThe 903rd Hospital of PLA,Hangzhou Zhejiang 310013;Department of Clinical Laboratory,Air Force Hangzhou Special Service Convalescent Center Convalescent Hospital AreaⅠ,Hangzhou Zhejiang 310013,China;Department of Traditional Chinese Medicine,The 903rd Hospital of PLA,Hangzhou Zhejiang 310013)
出处
《蚌埠医科大学学报》
2025年第2期234-238,共5页
Journal of Bengbu Medical University
基金
浙江省杭州市医药卫生科技规划项目(OO20190415)
浙江省医药卫生科技规划项目(2019331539)
浙江省自然科学基金项目(LGF20H200009)。
作者简介
周华君(1983-),男,副主任技师;通信作者:戴玉柱,副主任技师.E-mail:dyz5895@qq.com。
