牛副流感病毒3型RT—PCR检测方法的建立

摘要

With the primers based on the complete genome sequence of bovine parainfluenza virus 3（BPIV-3） in GenBank, a PCR method for bovine parainfluenza virus 3 detection was developed. The optimum condition for annealing temperature was 58.1℃, primer concentration was 1.0μmol/L. By using this method, the specific fragment of 425 bp was amplified from BPIV-3 template, but not from other common virus and bacteria, such as bovine respiratory syncytial virus, bovine viral diarrhoea virus, infectious bovine rhinotracheitis virus and swine fever virus, mycoplasma bovis strain HB0801, Escherichia coll., Pasteurella muhocida type A and Salmonella typhimurium. The sensitivity of this PCR method was 10-3 TCID50/0.1mL.%参照GenBank中公布的牛副流感病毒3型（Bovineparainfluenzavirus3，BPIV-3）全基因序列，针对BPIV-3特异性NP蛋白保守基因设计一对引物，建立了BPIV-3的RT—PCR诊断方法。其最佳扩增退火温度为58．1℃，引物浓度为1．0μmol／L。采用该方法扩增BPIV-3参考病毒．能扩增出425bp预期大小的特异性片段，而扩增牛病毒性腹泻／黏膜病病毒、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、大肠埃希氏菌、牛巴氏杆菌和沙门氏菌等常见病毒和细菌均呈阴性结果。对参考病毒进行梯度稀释检测，结果证明该法检测BPIV-3的灵敏度可达10^-3FCID50／0．1mL。