4种常见呼吸道病毒快速分子检测方法的建立

Establishment of a rapid molecular assay for detection of the four common respiratory viruses

目的建立一种基于液相芯片系统的4种常见呼吸道病毒快速分子检测方法,为有效防控其发生和流行提供有力手段。方法针对A型流感病毒(influenza A virus,FluA)、B型流感病毒(influenza B virus,FluB)、呼吸道合胞病毒A型(respiratory syncytial viruse types A,RSVA)和B型(respiratory syncytial viruse types B,RSVB)的保守区序列分别设计并合成了特异性引物、探针和通用引物,构建4种病原体阳性参比品,通过对扩增和杂交条件的优化,建立了靶序列富集多重PCR(target enriched multiplex PCR,Tem-PCR)和Luminex液相芯片(xMAP)技术相结合的检测系统,并对该检测系统进行了方法学评价。结果该检测方法可以特异性区分4种病毒的RNA靶标分子,检测下限均为10拷贝/μl。结论该种基于液相芯片系统的新方法灵敏度高、特异性好,为实现在临床呼吸道样品中同时快速检测4种常见呼吸道病毒提供了技术基础。

Objective To facilitate epidemiological viral monitoring,the Luminex x-MAP （Multiple Analysis Profiling ） sys- tem based Tem-PCR （Target enriched multiplex PCR） assay was developed to simuhaneously detect four different respiratory viruses including influenza A and B viruses, respiratory syncytial viruses types A and B. Methods Specific upstream and downstream primers, hybridization probes and super primers were designed based on the conserved sequences of available respiratory-virus sequence data- base. Recombinant plasmid and in vitro transcription RNA positive reference substances were established respectively. The detection systems were optimized and evaluated through specificity and sensitivity tests by the synthetic viral RNAs. Results There was no cross- reactivity among the four respiratory viral target RNAs. The all of detection limits for the four viral in intro-transcribed RNAs was 10 RNA copies. Conclusion We establish a sensitive and specific detection system suitable for detecting multiple respiratory viral RNAs for clinical specimens.