应用巢氏PCR方法快速检测猪鼻拭子样品中产毒素多杀性巴氏杆菌  

Detection of Toxigenic Pasteurella multocida Directly from Nasal Swabs with a Nested PCR Assay

作　　者：汤细彪[1] 吴斌[1] 刘国平[1] 杨明柳[1] 罗勇[1] 卢顺[1] 陈焕春[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室湖北省预防兽医学重点实验室,武汉430070

出　　处：《畜牧兽医学报》2007年第10期1083-1087,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(30471292)

摘　　要：根据产毒素多杀性巴氏杆菌的toxA基因序列,设计了2对特异性引物,扩增的片段大小分别为864和447 bp,从而建立了一种能直接从猪鼻拭子中快速检测产毒素多杀性巴氏杆菌的巢氏PCR方法。该方法能检出26CFU菌量以上的模板DNA,且不能从猪的其它7种常见病原菌扩增到特异性条带。通过对5个不同地区阳性猪群中采集的146份临床鼻拭子样品进行巢氏PCR检测和细菌分离鉴定,结果2种方法同时为阳性的样品有44份,同时为阴性的样品有97份,另有5份样品只有巢氏PCR检测为阳性,巢氏PCR检测与细菌分离鉴定的符合率为96.58%。试验表明:巢氏PCR方法能快速、灵敏地从猪鼻拭子样品中检出产毒素多杀性巴氏杆菌,适合临床进行大规模病原学检测,具有良好的应用前景。A nested polymerase chain reaction （N-PCR） assay for direct detection of toxigenic Pasteurella multocida （ T^＋ Pm） in nasal swab specimens collected from pigs was developed using 2 pairs of primers that were designed according to the published sequences of T^＋ Pm toxA, The size of amplicons were 864 and 447 bp, respectively. The assay could detect 26 CFU microorganisms. Mean while, no false amplification in detection of Escherichia coli, Streptococcus, Staphylococcus, Bordetella bronchiseptica , Nontoxigenic Pasteurella multocida , Haemophilus parasuis and Actinobacillus pleuopheumoniae could be observed. The nested PCR and bacterial isolatioh were employed simultaneously to examine 146 nasal swabs collected from 5 herds known to be infected with toxigenic Pasteurella multocida, 44 and 97 specimens were positive and negative for both isolation and nested PCR, respectively, 5 were positive for nested PCR only. The coincidence between bacterial isolation and nested PCR is 96.58%. These results indicated that the developed nested PCR might be a promising approach in clinical screening and detection of toxigenic Pasteurella multocida in a large number of swine swabs.

关 键 词：产毒素多杀性巴氏杆菌 toxA 鼻拭子 巢氏PCR 

分 类 号：S852.61[农业科学—基础兽医学]