| 摘要: |
| 根据禽流感病毒（AIV）的M基因序列设计合成了1对AIVM基因的通用诊断引物AMD1/AMD2，针对H5N1、H9N2两种亚型的M基因分型设计合成了2对分型诊断引物H5D1/H5D2，H9D1/H9D2。采用一步法多重RT-PCR技术建立了一种禽流感病毒快速分型诊断方法，并对其特异性和灵敏度进行验证。结果表明，该诊断方法速度快，一般只需要28～30 h即可鉴定出结果，比病毒分离鉴定（5～7 d）至少缩短4～6 d；特异性强，试验组53株AIV全部扩增出与预期同样长度的目的条带，与病毒分离及血清学鉴定方法的检测结果完全一致。对照组中新城疫病毒（NDV）、减蛋综合征病毒（EDS76V）、传染性支气管炎病毒（IBV）均未扩增出特异性基因条带,；灵敏度高，试验组将AIV接种SPF鸡胚，培养后的鸡胚尿囊液（AAF）进行10-2稀释后，仍可以检测到其中的AIV。 |
| 关键词: 禽流感病毒 M基因 HA基因 诊断方法 |
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| 基金项目:国家科技攻关项目(2002BA514A-15-3) |
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| Establishment and application of single-step multiplex RT-PCR for avian Influenza virus type and subtype detection |
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| Abstract: |
| The pair of primer AMDl and AMD2 were designed based on the strongly conservative region of the avian influenza virus(AIV)M gene,which hold specific property of A type AIV.The different subtype diagnosis primers H5D1/H5D2,and H9D1/H9D2 were respectively designed based on the HA gene of H5N1 and H9N2 subtype AIV.The single-step RT-PCR was established with three pairs of primers.The method of specificity and sensitivity were verified.The results showed that the specific elcctropho-resis stripes were 380 bp length for M gene,431 bp length for H5A gene and 387 bp length for H9A gene by RT-PCR with the three sets of primers.The PCR products showed the same length with the expected one.But the controlled NDV,EDS76V,IBV were not amplified by the RT-PCR.The shortest time of diagnosis was 28 hours,which was much shorter than other AIV diagnosis methods.The AIV could also be detected after those allantoic-amniotic fluid (AAF) were diluted 100 times.Fifty-three AIV and fifty samples were detected successfully,which were completely consistent with the results tested by serum methods. So the AIV detection via RT-PCR is very specific,sensitive,safe and time-saving.Thus this RT-PCR method is suited for application of AIV diagnosis in common laboratories. |
| Key words: Avian Influenza Virus M gene HA gene RT-PCR diagnosis |
