核酸探针检测鸡传染性支气管炎病毒方法的建立及应用  

Development and application of DIG labelled probe for detection of infectious bronchitis virus

作　　者：莫胜兰[1] 刘惠莉[2] 陆承平[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]上海市农业科学院畜牧兽医研究所,上海201106

出　　处：《中国兽医学报》2007年第2期168-171,共4页Chinese Journal of Veterinary Science

基　　金：上海市农委重点攻关资助项目(2004)

摘　　要：以地高辛(DIG)标记鸡传染性支气管炎病毒(IBV)pol基因的保守片段制成核酸探针,与IBV参考株、新城疫病毒、传染性法氏囊病病毒、禽流感病毒、正常鸡胚尿囊液及正常鸡肾组织等的RT-PCR产物进行斑点杂交,以检测探针的特异性,结果该探针仅与IBV毒株的RT-PCR产物杂交呈阳性,与对照病毒和组织的RT-PCR产物杂交呈阴性。敏感性试验显示,探针最低能检出约3.4pg的IBVRT-PCR产物。用该方法检测了38份疑似IBV临床病料,31份阳性;而用RT-PCR法扩增IBVS2基因确诊为阳性的只有29份。对人工接种IBV H52弱毒苗鸡咽喉和肛门拭子32份进行检测,检出15份阳性。结果表明,利用DIG探针检测IBV的RT-PCR产物,特异性和敏感性强,可重复,能克服RT-PCR非特异性反应和探针Northern杂交的不稳定性。The conserved region of avian infectious bronchitis virus(IBV) polymerase gene was labelled with digoxigenin as a DNA probe.The DIG-labelled fragment was tested by dot-blot hybridization with the RT-PCR prouduct of IBV reference strains and NDV,IBDV,AIV,the allantoic fluid of health egg,kidney tissue of health chicken for specificity. The result showed that only the IBV strains were positive.As little as 3.4 pg cDNA of IBV could be detected by the DIG-labelled probe.There were 38 IBV suspicious samples were detected by the two methods at the same time,31 out of 38 were positive by DIG-labelled probe;but only 29 of 38 were positive by RT-PCR to the S2 gene of IBV.Afterward,the DIG-labelled probe was used for detection and analysis of the 32 samples that collected from the pharynx and larynx or anus of the chicken which vaccinated with IBV H52 vaccine, with 15 out of which to be positive.It suggested that the DIG-labelled probe was superior in sensitivity,specificity and repetition.It would be able to avoid the non specificity of RT-PCR assay and the unsteadiness of probe Northern hybrization.

关 键 词：传染性支气管炎病毒 DIG探针 RT-PCR 检测 鸡 

分 类 号：S852.65[农业科学—基础兽医学]