摘要
根据已发表的猪胸膜肺炎放线杆菌APXIV毒素的基因序列 ,自行设计和合成了二对可扩增 448bp和 3 65bp目的片段的引物 ,成功的建立了检测APP的套式PCR方法。通过对猪肺疫巴氏杆菌、猪链球菌、大肠杆菌、猪嗜血杆菌、猪肺炎支原体和猪丹毒杆菌的DNA进行了PCR检测 ,结果均为阴性 ;对猪胸膜肺炎放线杆菌的 1、2、5、6、7、9国际标准血清型均扩增出 448bp和 3 65bp的特异性条带 ;检测的敏感度一步PCR可达到5 0 0个细菌 ,最低检出DNA浓度可达到0 .5 85ng/mL ;套式PCR可达到 5 0个细菌 ,最低检出DNA浓度可达到 5 8.5pg/mL。另外 ,对 5株从病猪体内分离的猪胸膜肺炎放线杆菌进行了检测 ,5株均成阳性反应 ;对 1 0只屠宰猪的肺脏分离物进行了检测 ,结果 1份为阳性。结果表明此法特异性和敏感性均很高 ,可做为猪传染性胸膜肺炎的快速诊断和流行病学调查的手段。
Two pairs of primers have been designed according to the sequence of apxIVA of Actinobacillus pleuropneumoniae(APP), which can specifically amplified the 448 bp and 365 bp fragments, respectively.And based on it we successfully developed the nested PCR method to detecting APP. We also tested the specificity and sensitivity of these PCRs and got the negative results by these PCRs with phylogenetically related species, i.e., Pasteurella multocida, Streptococcus suis, Escherichia coli, Haemophilus suis, Mycoplasma suipneumoniae and Erysipelothrix rhusiopathiae. Using these PCRs, positive amplifications were obtained with serotypes 1, 2, 5, 6, 7, 9 of APP reference strains. The sensitivity of single-step PCR is 500 bacteria, the minimum detecting concentration of DNA is 0.585ng/ml. For nested PCR, the sensitivity is 50 bacteria and 5.85×10 -8 g/ml. In addition, positive results were also obtained with 5 field strains and 1 out of 10 of lung isolates of slaughtered pigs. Due to the high specificity and sensitivity of the test, it can be served as a effective method for the rapid diagnosis and epidemiology research of APP.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2003年第4期305-309,共5页
Chinese Journal of Preventive Veterinary Medicine
