猪传染性胸膜肺炎放线杆菌PCR检测试剂盒的研制

根据GenBank中猪传染性胸膜肺炎放线杆菌apxⅣA基因序列,设计了1对引物,通过对PCR反应条件进行优化,研制了检测猪传染性胸膜肺炎放线杆菌的PCR试剂盒。该试剂盒扩增的阳性条带为600 bp,特异性与敏感性结果显示,该PCR检测试剂盒的最低核酸检测量为50 CFU/mL,而对金黄色葡萄球菌、链球菌、多杀性巴氏杆菌、鼠伤寒沙门氏菌、副猪嗜血杆菌、大肠杆菌的扩增结果均为阴性。-20℃至少可保存12个月,且重复性良好。应用该PCR试剂盒对41份临床样本进行了检测,其PCR检测结果与细菌学检测结果相一致。结果表明,猪传染性胸膜肺炎放线杆菌PCR检测试剂盒能够对APP临床样品进行快捷、灵敏、准确的检测。

Development of PCR Detection Kit for Actinobacillus pleuropheumoniae

According to the gene sequences in GenBank of APP apxⅣ A gene, one pairs of specific primer was designed for amplifying the specific fragments of apxⅣ A gene. After optimization of annealing temperature and primers concentrations, PCR was established for simultaneous detection of the APP. The specific bands of 600 bp was amplified. The sensitⅣity and specificity tests showed that the PCR was highly sensitⅣe in 50 CFU/mL. No band was amplified from Staphyloccocus aureus Rosenbach, Streptococcus, Pasteurella multocida, Salmonella typhimurium, Haemophilus parasuis, Escherichia coli by PCR, which could be stored over 12 months at -20 ℃. 41 clinical samples were detected by the PCR, which were coincided with their bacteriological test. The results revealed that the established PCR assay was sensitⅣe，specific and reproducible and it could be used to detect rapidly APP.