一步法复合RT-PCR鉴别ND强毒与AI病毒的研究  

MOLECULAR DETECTION OF VIRULENT NEWCASTLE DISEASE VIRUS AND AVIAN INFLUENZA VIRUS BY ONE-STEP DUPLEX RT-PCR

作　　者：黄兵[1] 马秀丽[2] 宋敏训[2] 王莉莉 李峰[2] 陈溥言[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点开放实验室,江苏南京210095 [2]山东省农业科学院家禽研究所,山东济南250023

出　　处：《西南农业大学学报（自然科学版）》2005年第6期881-884,共4页Journal of Southwest Agricultural University

基　　金：山东省科技攻关计划资助项目(031020101)

摘　　要：根据新城疫病毒(NDV)融合蛋白和禽流感病毒(AIV)核蛋白的核酸序列设计2对引物,对NDV和AIV的特异扩增片段分别为156 bp和219 bp,建立了同时检测强毒力NDV和AIV核酸的一步法复合RT-PCR。试验中未能检出弱毒力NDV,IBV,IBDV,REV,REOV及SPF鸡肌肉组织的RNA,从不同年代分离的16个NDV强毒株全为阳性而NDV弱毒株全为阴性。经验证,复合引物与单一引物的扩增效率一致,能够检出NDV和H5亚型AIV的最低病毒量分别为103.7EID50和103.9EID50。试验结果表明,此次建立的一步法复合RT-PCR特异、敏感,适用于强毒力NDV和AIV感染的快速鉴别。A simple one - step duplex reverse transcription polymerase chain reaction （ RT - PCR） was developed with two pairs of oligomlcleotide primer designed on the basis of the fusion protein of Newcastle disease virus （ NDV ） and the nucleoprotein of avian influenza virus （ AIV） , making possible the simultaneous detection of RNA of both NDV and AIV. The amplicons specific for NDV and AIV were 156 bp and 219 bp in length, respectively. Sixteen virulent NDV isolates in different periods were identified as positive while the others, including avirulent NDV, infectious bronchitis virus, infectious bursal disease virus and reticuloendotheliosis virus, Reovirus and the RNA from the chicken muscle, were negative. A 10^3.7EID50 of virulent NDV and 10^3.9EID50 of AIV subtype Hs could be discriminated by duplex primers in parallel with a single primer. These results suggest that this method is specific and sensitive, and is applicable for rapid differentiation of virulent NDV and AIV infection in chickens.

关 键 词：新城疫病毒 禽流感病毒 复合RT—PCR 分子检测 

分 类 号：S851.347.201[农业科学—预防兽医学]