猪链球菌、胸膜肺炎放线杆菌和副猪嗜血杆菌多重PCR检测方法的建立和应用

Development and application of a multiplex PCR for detection of Streptococcus suis,Actinobacillus pleuropneumoniae and Haemophilus parasuis

根据已发表的猪呼吸道疾病主要致病菌猪链球菌（SS）、胸膜肺炎放线杆菌（APP）和副猪嗜血杆菌（HPS）基因序列合成3对特异性引物,通过单一PCR和三重PCR体系及条件优化,建立这3种致病菌的三重PCR检测方法,并对200份临床表观健康猪鼻拭子样品用三重PCR方法进行检测。结果显示：建立的三重PCR方法能对同一样品中的SS、APP、HPS 3种病原菌的DNA模板进行扩增,无交叉反应;对猪体内常见的猪多杀性巴氏杆菌、猪大肠杆菌、猪放线杆菌、猪葡萄球菌、猪丹毒杆菌、猪沙门氏菌进行扩增,并无任何扩增产物;对SS、APP、HPS细菌纯培养物的检测敏感性分别为1×10^4CFU/ml、1×10^3CFU/ml、1×10^3CFU/ml;对人工模拟SS、APP、HPS感染组织样品的敏感性均为1×10^4CFU/ml。三重PCR方法对200份临床表观健康猪鼻拭子样品的检测结果显示：SS阳性160份,阳性率80.0%;APP阳性3份,阳性1.5%;HPS阳性45份,阳性22.5%。分别用细菌分离法和三重PCR法对30份临床病料进行检测,两者检测结果的符合率达到93%。建立的三重PCR可在4.5 h左右得到检测结果。

To achieve the purpose of simultaneously detecting three kinds of pathogenic bacteria in porcine respiratory diseases, Streptococcus suis （SS）, Actinobacillus pleuropneumoniae （APP） and Haemophilus parasuis （HPS）, three pairs of specific primers were synthesized from published SS, APP and HPS gene sequences, and the multiplex PCR method for SS, APP, and HPS was established. The multiplex PCR method could simultaneously amplify SS, APP and HPS in the same sample, without cross reaction. Pasteurella Multocida, Escherichia coli, A. suis, Staphylococcus aureus, Salmonella and swine erysipelas were detected by the multiplex PCR respectively and no specific products were amplified. The minimum detection concentrations of SS, APP and HPS were 1×10^4 CFU/ml, 1×10^3 CFU/ml and 1×10^3 CFU/ml, re-spectively. The sensitivity of the multiplex PCR method to artificially infected samples of SS, APP and HPS was 104 CFU/ml. Among 200 nasal swabs of apparently healthy pigs from Jiangsu province, the positive rates for SS, APP and HPS were 80-0% （160/200）, 1-5% （3/200） and 22-5% （45/200）, respectively. The total coincidence rate between the bacterial isolation and the multiplex PCR for 30 clinical diseased samples was 93% （28/30）. This multiplex PCR is a sen-sitive, specific and rapid method for the detection and identification of major bacteria in porcine respiratory disease complex, taking only 4-5 h to get the results.