基于SPR技术高通量呼吸道病毒检测方法研究

目的结合表面等离子共振(SPR)和基因芯片技术优势,针对9种常见呼吸道病毒[包括A型、B型流感病毒(Influ A,B),甲型H1N1,呼吸道融合病毒(RSV),副流感病毒1~3型(PIV1、2、3),腺病毒(ADV)以及引发严重急性呼吸综合征的冠状病毒(SARS)],构建特异性强、通量高的生物传感器方法。方法首先利用软件premier 5等在保守序列上设计相关病毒的特异性引物及探针;将所设计的9种相应呼吸道病毒的探针固定于化学修饰后的SPR芯片的特定区域,利用SPR技术实时监测探针与PCR产物的杂交过程,最后通过生物素与链霉亲和素系统实现信号的放大。结果设计的基因芯片可以高通量地检测9种呼吸道病毒,具有很好的检测特异性;芯片表面通过一定的再生条件后可以重复利用,避免芯片批间差对结果的影响;检测灵敏度都达到纳摩尔级。结论初步研究结果表明,利用SPR传感技术建立高通量检测呼吸道病毒的检测方法具有一定的实用性和可行性,有望成为快速、大规模、高通量筛查呼吸道病毒感染的手段,具有较好的应用前景。

Study on high-throughput respiratory tract virus detection method based SPR

Objective To develop a biosensor method with strong specificity and high‐throughput by combining with the surface plasmon resonance (SPR) and gene chip technique and aiming at 9 kinds of common respiratory tract viruses including influenza A and influenza B ,(Influ A ,B) ,H1N1 ,respiratory syncytial virus (RSV) ,parainfluenza virus 1 -3 (PIV1 -3) ,adenovirus (ADV) and coronavirus (SARS) leading to severe acute respiratory syndrome .Methods Firstly the software primer 5 was used to design the specific primer and probe of related viruses in the conserved sequence ;the designed nine kinds of corresponding respiratory virus probes were immobilized in the specific region of SPR chip after chemical modification .The SPR technique was applied to conduct the real time monitoring the hybridization process of the probe with the PCR products .Finally the signal amplification was realized by the biotin and streptavidin system .Results The designed gene chip could detect 9 kinds of respiratory tract viruses by high‐throughput with better detection specificity ;the chip surface could be reutilized after certain regeneration condition ,which avoided the influence of intra‐batch difference on the results ;the detection sensitivity reached the nanomole level .Conclusion The prelimi‐nary study results demonstrate that using the SPR biosensor technique to establish a high‐‐throughput detection of respiratory tract viruses has some practicability and feasibility ,and is expected to become a rapid ,large scale and high‐ throughput measure for screening respiratory tract viruses with good application prospect .