一种基于液相基因芯片技术对甲型和乙型流感病毒分型检测方法的建立

Development of a method for detecting type influenza virus A and B based on liquid-phase gene chip technology

目的建立能够对甲型流感病毒(influenza virus A,INFA)和乙型流感病毒(influenza virus B,INFB)进行区分的液相基因芯片技术,为甲型和乙型流感病毒的分型提供一种高通量、高灵敏度的检测和鉴定方法。方法从NCBI的Gen Bank下载具有代表性的流感病毒基因组序列,利用生物信息学软件Bio Edit进行序列比对,分别设计针对INFA和INFB的简并引物和特异性探针,将设计好的引物和探针与Gen Bank中所有的基因序列进行比对,以验证其特异性。经探针合成、悬浮液相芯片制备和检测条件的优化获得用于甲型和乙型流感病毒分型的液相基因芯片。结果所检测INFA和INFB均可获得特异性杂交信号,非特异性杂交信号不明显,将2种病毒混合,模拟混合病毒感染也可以得到相应的特异性杂交信号。灵敏度评估结果表明INFA的检测灵敏度为1~10空斑形成单位(plaque forming unit,pfu),而INFB为10~100 pfu。将本方法用于检测11份临床样本,其结果与荧光定量分型逆转录聚合酶链反应(reverse transcriptase-polymerase chain reaction,RT-PCR)检测方法的结果一致。结论本研究初步建立了INFA和INFB的液相基因芯片检测技术,可用于流感病毒的初步筛查和鉴定,并且可以进行混合感染样本的检测,为流感病毒的分型和鉴定提供了一种新的手段。

Objective To develop a microbead-based liquid assay for rapid and high throughput detection and the typing of influenza virus A and B（INFA, INFB）. Methods Representative sequences has been downloaded from the Gen Bank database and aligned with Bio Edit software. Specific primers and probes have been designed based on conserved sequences. The specificity of the primers and probes has been tested by blast of them with all the gene sequences in the Gen Bank database. The microbead-based liquid assay was developed by covalent linkage of the probes and microbeads. Results Specific signals of mean fluorescent intensity have been observed in all of the tested samples. Very low signals have been obtain for non-specific samples. Mixture of both influenza virus A and B can obtain their positive signal respectively. The sensitivity of the method for INFA is 1-10 pfu and 10-100 pfu for INFB. 11 clinical samples were detected using this method and the results were consistent with the detection results of real time reverse transcriptase-polymerase chain reaction（RT-PCR）. Conclusion The microbead-based liquid assay developed in this study for the typing of influenza virus A and B can be used for the detection and typing of influenza viruses. The assay provides a new method for the detection of the viruses.