ARV、REV与ALV三重RT-PCR检测方法的建立

Establishment and application of thistriplex RT-PCR for ARV,REV and ALV

根椐GenBank中已发表的禽呼肠孤病毒(ARV)、禽网状内皮增生病病毒(REV)、禽白血病病毒(ALV)等3种病毒基因组序列,设计了3对分别与ARV、REV和ALV某段基因序列互补的引物。在建立各病毒单项RT-PCR技术的基础上,优化三重RT-PCR反应条件,建立了3种病毒的三重RT-PCR技术。结果表明,用这3对引物对同一样品中的ARV、REV、ALV核酸模板进行三重RT-PCR扩增,可同时扩增ARV的247bp,ALV的675bp,REV的467bp的特异性片段,而对其他6种禽病病原的PCR扩增结果均为阴性。敏感性测定结果表明,该三重RT-PCR技术能检出10pg的ALV、1pg的ARV和10pg的REV模板。用42份临床病料对本研究多重RT-PCR技术和单项RT-PCR技术进行对比验证,结果显示,两者的总符合率为92%以上。表明建立的多重RT-PCR检测方法,具有特异、快速、准确的特点,可用于对这3种病毒的同时检测和鉴别诊断。

A thistriplex RT-PCR was developed to simultaneously detect three pathogens of avian reovirus（ARV）,reticuloendotheliosis virus（REV） and avian leukosis virus subgroup J（ALV-J） in this study.Three sets of specific primers were designed according to the sequences of ARV,REV and ALV-J in the GenBank.By using three pairs of virus specific primers,three RT-PCR assay were established to amplify the conservative regions of the three viruses,respectively.Consequently,a thistriplex RT-PCR method to detect the three viruses in one tube was developed.The thistriplex RT-PCR system would amplify a 247 bp fragment for ARV,a 467 bp for REV and a 675 bp for ALV simultaneously or separately in the samples,depending on its infection status.But not specific band was amplified from other six avian pathogenic viruses.As little as 10 pg of REV,1 pg of ARV and 10 pg of ALV RNA were detected using gel electrophoresis in thistriplex RT-PCR.To evaluate the thistriplex RT-PCR,42 clinical samples were comparatively detected.The data showed that the thistriplex RT-PCR method was 92% coincidence with the single RT-PCR,and it can be used for this three viruses detection and differential diagnosis.