一种基于NASBA技术的新型快速肠道病毒检测方法的应用研究

Development of a new method based on NASBA for enterovirus fast detection

目的:基于核酸等温扩增技术(nucleic acid sequence-based amplification,NASBA)荧光分子信标探针检测技术,进行肠道病毒的快速检测与辅助诊断。方法:根据Genebank上肠道病毒5′端非编码区序列,设计特异性引物与捕获探针,应用纳米技术对商品化磁珠进行偶联,NASBA方法进行扩增,NucliSens读数仪检测,荧光定量RT-PCR方法进行验证,同时验证NASBA方法的特异性、灵敏度和重复性。结果:该方法对肠道病毒具有高度特异性,与RSV等呼吸道相关病毒均无交叉反应,反应体系具有很高的稳定性。结论:本研究应用的肠道病毒NASBA检测方法特异、灵敏、快速简便,适用于肠道病毒的日常监测和爆发疫情的应急诊断。

Objective： To develop a new nucleic acid sequence-based amplification（NASBA） method which is based on fluorescence probe for the detection of enterovirus and clinic diagnosis.Methods： The special primers and probes were designed based on the nucleic acid sequences of relative enterovirus in Gene bank.The commercial immunomagectic beads were conjugated with the primers and probes using nanotechnology.The conjugated products were amplified by NASBA and sequentially detected by NucliSens reader.The results were validated by real time-PCR.The sensitivity,repetition and specificity of the method were evaluated respectively.Results： The method could detect enterovirus with high specificity and stability as well as no interactions with other respiratory virus such as RSV.Conclusion： The new NASBA method is convenience,fast,sensitive and specific for enterovirus detection,which could be applied for regular surveillance and urgent investigation of outbreaks of enterovirus in the near future.