牛副流感3型病毒、牛传染性鼻气管炎病毒、牛病毒性腹泻病毒和牛支原体多重PCR检测方法的建立

Establishment of a multiplex PCR for detection of bovine parainfluenza virus 3,infectious bovine rhinotracheitis virus,bovine viral diarrhea virus and Mycoplasma bovis

为建立检测牛副流感3型病毒(BPIV3)、牛传染性鼻气管炎病毒(IBRV)、牛病毒性腹泻病毒(BVDV)和牛支原体(M.bovis)的多重PCR检测方法,本研究根据GenBank中登录的BPIV3的HN基因、IBRV的g C基因、BVDV的5'UTR和M.bovis的oppD/F基因序列设计特异性引物,通过优化反应条件建立了一种可以同时检测以上4种病原的多重PCR方法。结果显示,该方法对牛呼吸道合胞体病毒、小反刍兽疫病毒、牛布鲁氏菌、羊布鲁氏菌、牛源多杀巴氏杆菌A型和B型无特异性扩增,对BPIV3和BVDV的cDNA最低检测量分别为100 pg和1 ng,对IBRV和M.bovis的DNA最低检测量分别为10 pg和100 pg,具有较高的特异性和灵敏性。对临床33份鼻拭子样品和12份牛肺样品的检测结果与已发表文献中PCR方法的检测结果一致。本研究建立的多重PCR检测方法可同时对BPIV3、IBRV、BVDV和M.bovis进行检测,为这4种病原的诊断和检测提供了一种实用、便捷的方法。

To establish a detection method for simultaneously detecting bovine parainfiuenza virus 3 （BPIV3）, infectious bovine rhinotracheitis virus （IBRV）, bovine viral diarrhea virus （BVDV） and Mycoplasma bovis, the multiplex PCR （mPCR） assay was developed with a set of specific primers designed based on HN, gC, 5＇UTR, oppD/F genes of BPW3, IBRV, BVDV and M. bovis, respectively. Meanwhile, the primers ratio and annealing temperature were optimized. The detection results showed that the mPCR was able to simultaneously amplify the specific fragments of 510bp for BPIV3, 308bp for IBRV, 215bp for BVDV and 78bp for M.bovis, which had no any amplification for BRSV, PPRV, B.melitensis, B.abortus, bovine P.multocida serotype A and bovine P.multocida serotype B. The detection sensitivity of the mPCR for the DNA/cDNA of BP1V3, IBRV, BVDV and M.bovi were 100pg, 10pg, lng and 100pg, respectively. Additionally, the established mPCR assay was compared with traditional single PCR for 33 nose swab samples and 12 bovine lung samples detections, and the coincidence rate was 100%. These results demonstrated that the established mPCR was high effectively and sensitivity in detecting the four pathogenic microorganisms.