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中文摘要：
参照GenBank中登录的牛副流感病毒3型（BPIV-3）和牛病毒性腹泻病毒（BVDV）全基因序列,分别针对BPIV3特异性NP蛋白保守基因和BVDV保守区段E2基因设计2对引物,经优化反应条件建立了快速鉴别BPIV-3和BVDV的双重RT-PCR诊断方法。最佳扩增条件为94℃30s,56.2℃30s,72℃1min,循环30次;72℃延伸5min,16℃10min;BVDV引物浓度为1.0μmol/L,BPIV-3引物浓度为0.5μmol/L。采用该方法检测BPIV-3和BVDV参考病毒株,能同时扩增出预期为425bp和294bp大小的特异性片段,而扩增牛传染性鼻气管炎病毒、牛合胞体病毒、猪瘟病毒以及牛支原体、致病性大肠埃希菌、多杀性巴氏杆菌A型、化脓隐秘杆菌和鼠伤寒沙门菌等均呈阴性反应。对参考病毒株进行梯度稀释检测,结果证明该方法检测BPIV-3的灵敏度可达10^-3 TCID50/0.1mL,而BVDV的灵敏度达102 TCID50/0.1mL。
英文摘要：
With primers specific to nucleotide sequences of bovine parainfluenza virus 3（BPIV-3） NP gene and bovine viral diarrhea virus（BVDV） E2 gene from GenBank,the duplex RT-PCR method for bovine parainfluenza virus 3 and bovine viral diarrhea virus detection was developed.The optimum condition is as followings： 94 ℃30 s,56.2 ℃30 s,72 ℃1 min for 30 cycle,72 ℃ 5 min,16 ℃ 10 min;BVDV primer concentration of 1 μmol /L,BPIV-3 primer concentration of 0.5 μmol /L.By using this method,the specific fragment of 425 bp was amplified from BPIV-3 samples while that of 294 bp from BVDV.None target fragments were amplified from bovine respiratory syncytial virus（BRSV）,infectious bovine rhinotracheitis virus（IBRV） and classical swine fever virus（CSFV）,Mycoplasma bovis strain HB0801,Esche richia coli,Pasteurella coli type A and rat typhoid Salmonella.By this method,a minimal sample of 10^-3 TCID50 /0.1 mL could be detected from BPI^-3,minimal sample of 10^2 TCID50 /0.1 mL could be detected from BVDV.
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