猪瘟病毒、猪繁殖与呼吸障碍综合征病毒和乙型脑炎病毒多重RT-PCR检测方法的建立  

Development of Multiplex RT-PCR to Detect Classical Swine Fever Virus(CSFV),Porcine Reproductive and Respiratory Syndrome Virus(PRRSV) and Japanese Encephalitis Virus(JEV)

作　　者：刘辉[1] 熊金凤[1] 邹浩勇[1] 陈杨[1] 杨维红[1] 何启盖[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室/预防兽医学湖北省重点实验室,武汉430070

出　　处：《华中农业大学学报》2008年第1期12-18,共7页Journal of Huazhong Agricultural University

基　　金：国家科技支撑计划(2006BAD06A12);华中农业大学留学回国人员科研启动基金资助

摘　　要：根据猪瘟病毒(CSFV)的E2基因保守序列、猪繁殖与呼吸障碍综合征病毒(PRRSV)的ORF7基因及部分ORF6和3-UTR基因序列和猪乙型脑炎病毒(JEV)的E基因保守序列,设计了3对引物,扩增大小分别为288 bp4、30 bp和1 015 bp目的片段。以纯培养病毒抽提RNA,制备cDNA模板,利用3对引物,通过条件的优化,建立了同时检测这3种病毒的多重RT-PCR。CSFV、PRRSV和JEV的RNA最小检出量分别为0.270 ng、0.049 ng、0.067 ng,其它的RNA病毒如TGEV以及DNA病毒如PPV、PrV、PCV-2检测结果为阴性。以-βactin为对照,利用本方法检测了174份临床样品(血清、肺脏和胎儿),其结果为:PRRSV、CSFV和JEV的阳性率分别为30.4%、4.6%和1.7%;CSFV和PRRSV混合感染阳性率为1.7%。同时对临床样品中CSFV、JEV和PRRSV阳性PCR产物进行测序分析,发现CSFV、PRRSV和JEV的PCR产物序列和NCBI数据库中目标基因的同源性分别为92%、90%和89%,这证实了多重PCR的阳性结果为上述3种病毒。结果证明,所建立的多重RT-PCR可用于临床检测。This paper described a multiplex RT-PCR to rapidly diagnose the co-infection in samples from pig with reproductive failure symptoms.Three sets of primers were designed,respectively,targeting to the conserved sequences in E2 gene of classical swine fever virus（CSFV）,a domain encompassing partial ORF6,ORF7 and 3-terminus UTR of porcine reproductive and respiratory syndrome virus（PRRSV） and E gene of Japanese encephalitis virus（JEV）.The expected sizes of RT-PCR amplicons were 288 bp for CSFV,430 bp for PRRSV and 1 015 bp for JEV,respectively.RNA was extracted from pure culture of the viruses,followed by the synthesis of cDNA that was used as templates for method development.Through optimization by using three sets primers simultaneously,the detection limit of RNA template in the RT-PCR was 0.27 ng for CSFV,0.049 ng for PRRSV and 0.067 for JEV.No false positive results were produced in the case of transmissible gastroenteritis virus（TGEV）,porcine parvovirus（PPV）,pseudorabies virus（PrV） and porcine circovirus type 2（PCV-2）.There were 174 samples,including sera,fetuses and lung tissues which were collected from the pigs that mainly presented high fever,were subjected to our in-house RT-PCR.PRRSV was detected in these samples with higher detection rate than CSFV and JEV.Among 174 clinical samples,PRRSV-positive percentage was 30.4,CSFV-positive percentage was 4.6,JEV-positive percentage was 1.7,co-infection of CSFV and PRRSV percentage was 1.7.In order to confirm the reliability of the multiplex PCR,we selected PCR products of CSFV-positive JEV-postive and PRRSV-positive respectively that detected clinical samples by multiplex PCR.After sequences analysis and BLAST,it is showed that homologies between PCR products of CSFV,PRRSV,JEV and target genes sequences were 92%,90% and 89%,respectively.Those results proved that multiplex RT-PCR have Applied value in practices.

关 键 词：多重RT-PCR 猪瘟病毒 猪繁殖与呼吸障碍综合征病毒 乙型脑炎病毒 

分 类 号：S854.43[农业科学—临床兽医学]