【摘要】 【目的】建立基于鸡传染性支气管炎病毒(IBV)S1基因的反转录-复合PCR方法,为IBV流行毒株和呼吸型疫苗株的分型鉴定提供技术支持。【方法】根据IBVN基因序列设计合成1对特异性通用引物,建立IBV的分子生物学检测方法,用以对IBV进行检测。通过对GenBank上公布的IBVS1基因全序列的分析比对,找出呼吸型疫苗株和地方肾型流行毒株相对保守的差异序列,设计合成2对引物,建立可以对这2类IBV毒株进行鉴别的反转录-复合PCR方法,同时对该方法的敏感性和重复性进行检测,并对该方法进行了实际应用。【结果】建立了区别IBV肾型毒株和呼吸型毒株的分型方法,该方法敏感性好,能检出经106稀释后的基因组RNA,且具有可重复性;临床应用显示,该方法对呼吸型毒株和近年来的地方肾型分离株具有较好的分型效果,而对较早时期的肾型分离株(YI株和澳大利亚T株)的扩增结果为阴性;经鉴定,近年来感染鸡群并引起高死亡率的分离毒株主要为肾型IBV。【结论】建立了区别IBV肾型毒株和呼吸型毒株的分型方法,该方法特异性强、敏感性高、重复性好,能将我国地方流行的肾型IBV毒株与呼吸型疫苗株完全区分开来。更多还原

【Abstract】 【Objective】Based on the S1 gene of IBV,the reverse transcription-multiple PCR methods were established to differentiate the epidemic Nephropathogenic strains and respiratory-type vaccine strains of IBV.【Method】 According to the conservative nucleocapsid gene sequences of avian infectious bronchitis virus(IBV),a pair of specific primers was designed and synthesized.And on this basis the molecular biological detection methods of IBV were established to assay the IBV isolates.At the same time,by analyzing the complete sequences of the IBV S1 gene published on GenBank,the relatively conservative differential sequences of respiratory-type vaccine strain and local nephropathogenic epidemic strains were found out.Two pairs of primers were designed and synthesized and the reverse transcription-multiple PCR methods were established to type for the two serotypes of IBV.Simultaneously,the method whose sensitivity and reproducibility had been tested was applied to clinical detection.【Result】 The typing method was established successfully.The genome RNA diluted to 10-6 could still be amplified effectively using this method.The results showed that:the methods could distinguish respiratory-type strains from the local nephropathogenic strains isolated in recent years better;the nephropathogenic strains(YI strain and the Australian T strain) isolated in the earlier period were not amplified effectively;in recent years,the strains that could infect chickens and cause high mortality mainly were nephropathogenic IBV.【Conclusion】 The typing method distinguishing local nephropathogenic epidemic strains from respiratory-type vaccine strains of IBV was established.The method has characteristics of strong specificity,high sensitivity and good repetition.And the method can distinguish completely the local nephropathogenic IBV from the respiratory-type IBV vaccine strains,and supply reference for detection and vaccine options of IB.更多还原