禽流感病毒分型基因芯片的研制

Oligonucleotide microarray for subtyping avian influenza virus

摘要:

【目的】禽流感病毒是一种全球重要的人和动物呼吸道病病原，快速确定其不同亚型对于全球流感监测具有重要的意义。本研究意在研制一种可同时鉴定禽流感病毒所有亚型的方法。【方法】根据GenBank上已发表的禽流感病毒不同亚型（16个HA亚型和9个NA亚型）的基因序列，设计合成了25对特异性引物和1对通用引物，然后以各亚型病毒的参考株RNA作为模板，建立扩增不同亚型的多重RT-PCR方法。参考各亚型病毒靶cDNAs区域的保守序列设计了52条亚型特异的探针，进而利用扩增的各亚型病毒的靶cDNAs对其特异性进行评价。在此基础上，将设计好的探针点制到处理好的玻片上，制备了禽流感病毒分型鉴定基因芯片，结合所建立的扩增不同亚型的多重RT-PCR方法，开发了禽流感病毒亚型鉴定基因芯片试剂。利用收集自49个地区的2653份标本对其特异性和敏感性进行了初步评价。【结果】用于评价的各亚型参考毒株均出现良好的特异性杂交信号，检测的敏感度可达2.47 PFU/mL或2.5 ng靶DNA片段，而且与禽类常见的IBV、NDV等 6种病毒均无交叉反应。【结论】证明该病毒分型基因芯片具有良好的特异性、敏感性。

Abstract:

[Objective] Avian influenza viruses are important human and animal respiratory pathogens and rapid diagnosis of novel emerging avian influenza viruses is vital for effective global influenza surveillance. We developed an oligonucleotide microarray-based method for subtyping all avian influenza virus (16 HA and 9 NA subtypes). [Methods] In total 25 pairs of primers specific for different subtypes and 1 pair of universal primers were carefully designed based on the genomic sequences of influenza A viruses retrieved from GenBank database. Several multiplex RT-PCR methods were then developed, and the target cDNAs of 25 subtype viruses were amplified by RT-PCR or overlapping PCR for evaluating the microarray. Further 52 oligonucleotide probes specific for all 25 subtype viruses were designed according to published gene sequences of avian influenza viruses in amplified target cDNAs domains, and a microarray for subtyping influenza A virus was developed. Then its specificity and sensitivity were validated by using different subtype strains and 2653 sam-ples from 49 different areas. [Results] The results showed that all the subtypes of influenza virus could be identified si-multaneously on this microarray with high sensitivity, which could reach to 2.47 pfu/mL virus or 2.5 ng target DNA. Fur-thermore, there was no cross reaction with other avian respiratory virus. [Conclusion] An oligonucleotide microar-ray-based strategy for detection of avian influenza viruses has been developed. Such a diagnostic microarray will be useful in discovering and identifying all subtypes of avian influenza virus.