猪繁殖障碍性疫病常见病毒多重PCR诊断方法的建立及初步应用  

Establishment and preliminary application of a mutiplex PCR method for detection viruses induced reproductive failure of swine

作　　者：关男 卢赫[1] 费东亮[1] 王书全[1] 

机构地区：[1]辽宁医学院畜牧兽医学院,辽宁锦州121001

出　　处：《中国兽医学报》2016年第1期5-11,共7页Chinese Journal of Veterinary Science

基　　金：辽宁省教育厅科研项目(L2014327)

摘　　要：根据GenBank登录的猪细小病毒病(PPV)、猪伪狂犬病病毒(PRV)、猪圆环病毒病2型(PCV2)、猪繁殖与呼吸综合征病毒欧洲分离株LV(PRRSLV)和美洲分离株VR(PRRSVR)等5种病毒基因序列设计合成5对特异性引物。通过优化反应条件,建立了同时检测5种病毒混合感染的多重PCR方法,对其特异性、敏感性、重复性进行检测;并将其应用于临床病料检验。结果显示:多重PCR方法中,当PPV、PRV、PCV2、PRRSVR和PRRSLV引物浓度为1.0、0.5、0.5、1.5、1.5μmol/L、退火温度为60℃时,扩增效果最佳,其扩增出特异性目的片段分别为142、331、547、705、1 367bp,最低检测量分别为155、45.2、17.2、14.2、43.6pg,在不同时间对相同样本重复检验6次,结果一致;对临床收集的125份疑似猪病料样品进行多重PCR技术和单一PCR技术进行对比检测,总符合率在97.9%以上,PPV、PRV、PCV2、PRRSLV和PRRSVR的阳性率分别为31.20%、36.8%、34.4%、1.6%和40.8%,混合感染率分别为16.8%、28.8%、23.2%、1.6%和30.4%。结果表明:本试验建立的多重PCR方法具有敏感性高、特异性强、稳定性好,可检测PPV、PRV、PCV2、PRRSVR和PRRSLV的混合感染。A multiplex polymerase chain reaction assay was developed for the clinical detection of porcine parvovirus virus（PPV）,Porcine pseudorabies virus （PRV）,porcine circovirus 2 （PCV2）, porcine reproductive and respiratory syndrome virus isolates LV （PRRSLV） in Europe and iso- lates VR （PRRSVR） in America which caused porcine reproductive barrier. We designed 5 pairs of specific primers, according to PPV, PRV, PCV2, PRRSLV and PRRSVR gene sequence from GenBank. By optimizing reaction conditions, the multiplex PCR reaction was developed. The speci- ficity,sensitivity and repeatability of the multiplex PCR were tested. All target fragments were well amplified, when the primer concentration of PPV, PRV, PCV2 ,PRRSVR and PRRSLV were 1.0,0.5,0.5,1.5 and 1.5 /~mol/L respectively, and the annealing temperature was 60~C. The spe- cificity test showed that fragments of 142,331,547,705 and 1 367 bp were amplified from the ge- nomic DNA （cDNA） of PPV, PRV, PCV2, PRRSVR and PRRSLV, respectively. The minimum detectable amounts were 155 pg for PPV,45.2 pg for PRV,17.2 pg for PCV2,14.2 pg for PRRS- VR and 43.6pg for PRRSLV. The results of 6 replication test for each sample at different time were consistent indicating that multiplex PCR had a good repeatability. The 125 clinical samples were detected by multiplex PCR and single PCR, both PCR methods shared 97.9% coincidence,a- mong these samples, the positive rate of PPV, PRV, PCV2, PRRSLV and PRRSVR were 31.20%,36. 8%,32. 4%,1. 6% and 40. 8% respectively, the co-infection rate were 16. 8%,28.8% ,23.2%,1. 6%and 30.4% respectively. high sensitivity, specificity and stability, which PPV, PRV, PCV2, PRRSVR and PRRSLV. These results indicated that multiplex PCR had can be used for detection of mixed infection of

关 键 词：猪繁殖障碍性疾病 多重PCR 建立 应用 

分 类 号：S852.65[农业科学—基础兽医学] S855[农业科学—兽医学]