猪繁殖与呼吸综合征病毒和日本脑炎病毒二联基因芯片的构建与应用  

Development and application of a microarray assay for simultaneous detection of porcine reproductive and respiratory syndrome virus and Japanese encephalitis virus

作　　者：欧阳达[1] 姜来生[1] 黄小波[1] 张仙[1] 常晓霞[1] 杨国林[1] 马锐[1] 滑翔 赵玉佳[1] 曹三杰[1] 伍锐[1] 文翼平[1] 文心田[1] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014

出　　处：《中国兽医科学》2015年第5期509-516,共8页Chinese Veterinary Science

基　　金：公益性行业(农业)科研专项(201203056)

摘　　要：针对靶基因库中猪繁殖与呼吸综合征病毒(PRRSV)的重组质粒T/PRRS-PS9、T/Y11和日本脑炎病毒(JEV)的重组质粒T/PRM/M、T/JEV-C,设计4对特异性引物,采用PCR扩增获得4个靶基因片段,并用PCR标记Cy3探针,建立了一种检测PRRSV和JEV的芯片方法。特异性试验结果显示,PRRSV、JEV之间无交叉杂交,猪瘟病毒、猪细小病毒、伪狂犬病病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒与PRRSV-JEV基因芯片无杂交反应。灵敏性试验结果显示,50μL杂交体系中探针DNA的质量浓度为0.295pg/μL时仍为杂交阳性。对从猪场收集的31份样品进行处理并用基因芯片检测,PRRSV的检出率为12.9%(4/31),JEV的检出率为9.68%(3/31),PRRSV与JEV混合感染的检出率为0(0/31)。此方法的结果与PCR的检测结果完全符合。结果表明,所建立的二联基因芯片有望成为一种快速鉴别PRRSV与JEV的新方法。In order to establish a new microarray-based assay for detecting and identifying porcine reproductive and respiratory syndrome virus(PRRSV)and Japanese encephalitis virus(JEV)using Cy3-labelled probes,4pairs primers were designed based on plasmid cloning bacteria vector T/PRRS-PS9,T/Y11,T/PRM/M and T/JEV-C.In result,there was not cross-hybrid between target genes and probes of PRRSV and JEV.And the probes of PRRSV and JEV could not hybrid with classical swine fever virus,porcine parvovirus,pseudorabies virus,transmissible gastroenteritis virus of swine,porcine epidemic diarrhea virus,respectively.The microarray assay had a sensitivity when the concentration of JEV cDNA was0.295pg/μL.Thirty-one clinical samples from pigs were tested for PRRSV and JEV by the microarray and PCR method,of which 12.9%(4/31)were positive for PRRSV,9.68%(3/31)for JEV and 0(0/31)for both,the microarray test result agreed well with the PCR result.The results indicated that the microarray has a high sensibility for the identification and detection of PRRSV and JEV.

关 键 词：猪繁殖与呼吸综合征病毒 日本脑炎病毒 基因芯片 繁殖障碍 

分 类 号：S852.651[农业科学—基础兽医学]