RT-PCR和Digoxigenin标记的核酸探针检测鸡传染性支气管炎病毒试验研究  

A Study on RT PCR and Digoxigenin labeled DNA Probe for Detection of Chichen Infectious Bronchtis Virus

作　　者：王泽霖[1] 王丽[1] 阎若潜 刘素云 菅复春[1] 

机构地区：[1]河南农业大学牧医工程学院

出　　处：《河南农业大学学报》1997年第4期304-309,共6页Journal of Henan Agricultural University

基　　金：河南省科技攻关资助

摘　　要：用２对已发表的引物和１对自行设计的引物对同一ＩＢＶＨ１２０株进行ＲＴ－ＰＣＲ，分别获得了Ｓ１基因上与引物设计相一致的１７２０ｂｐ，２２８ｂｐ，６０２ｂｐ，的扩增片段。用自行设计的引物对７个毒株（Ｈ１２０，Ｈ５２，Ｍ４１，Ｃｏｎｎ，Ｇｒａｙ，Ｔ，Ｈｏｌｔｅ）和５个分离株（宜毒，上毒，云毒，ＨＫ，１１８）的含毒尿囊液或纯化病毒进行ＲＴ－ＰＣＲ。结果除Ｈｏｌｔｅ株，２个分离株（宜毒，云毒）外，其余均被成功地扩增出６０２ｂｐ的片段。将ＩＢＶＨ１２０株０６ｋｂＰＣＲ产物用Ｄｉｇｏｘｉｇｅｎｉｎ标记为探针，分别与上述各ＩＢＶ毒株的核酸及其扩增产物进行核酸杂交，均呈现阳性反应，而该探针不与ＩＢＤＶ和ＮＤＶ的ＤＮＡ，ＥＤＳ－７６病毒的ＤＮＡ及正常鸡胚尿囊液抽提物反应。ＲＴ－ＰＣＲ和核酸杂交技术提供了直接从尿囊液中快速检测出病毒，作为诊断ＩＢＶ的新方法。Reverse transcription (RT) of viral RNA and polymerase chain reaction (PCR) were used to amplify the IBV H 120 viral genome using two pairs of primers previously published and a pair of self-designed primer.Three special PCR fragments of 1 720bp,228bp and 602bp corresponding to the threeprimers were obtained respectively.The PCR products which contained the S 1 glycoprotein gene of IBV were of the expected size.The IBV viral RNA extracted from the allantoic fluid of infected embronating eggs or purified virus from 7 different IBV reference strains and 5 field strains were amplified by RT-PCR using the self-designed primer.Except one reference strain and 2 field strains,all the other strains were successfully amplified.The PCR products appeared to be approximate 600bp,the expected size.The purified DNA fragment of approximate 600bp of IBV H 120 strain was labeled by digoxigenin as a DNA probe.The dot-hybridization assay with the DNA probe was used to detect the PCR products and RNA from all the IBV reference and field strains.All the IBV strains were positive.No hybridization was detected when RNA template from allantoic fluid of uninoculated eggs or from other virus including NDV,IBDV and EDS -76.The RT-PCR and dot-hybridization assay provide a new rapid method for detection and diagonosis of the IBV antigen from the allanotic fluid of chicken embronating eggs.

关 键 词：传染性 支气管炎病毒 鸡病 检测 核酸探针 

分 类 号：S858.315.3[农业科学—临床兽医学]