Register Karen B, DeJong Keith D
Respiratory Diseases of Livestock Research Unit, USDA/Agricultural Research Service/National Animal Disease Center, Ames, IA 50010, USA.
Vet Microbiol. 2006 Oct 31;117(2-4):201-10. doi: 10.1016/j.vetmic.2006.05.003. Epub 2006 May 16.
Bordetella bronchiseptica and Pasteurella multocida are etiologic agents of progressive atrophic rhinitis (PAR) and bronchopneumonia in swine. Only dermonecrotic toxin-producing strains of P. multocida play a role in atrophic rhinitis while both toxigenic and nontoxigenic strains have been associated with pneumonia. Monitoring and investigation of outbreaks involving these bacteria require sensitive and accurate identification and reliable determination of the toxigenic status of P. multocida isolates. In the present study, we report the development, optimization, and performance characteristics of a multiplex PCR assay for simultaneous amplification of up to three different targets, one common to all P. multocida strains, one found only in toxigenic P. multocida strains, and one common to B. bronchiseptica strains. Based on analysis of 94 P. multocida isolates (31 toxigenic) and 126 B. bronchiseptica isolates assay sensitivity is 100% for all amplicons. Evaluation of 22 isolates of other bacterial genera and species commonly found in the swine respiratory tract demonstrated a specificity of 100% for all gene targets. The limit of detection for simultaneous amplification of all targets is 1-10pg of DNA per target, corresponding to a few hundred genomes or less. Amplicon mobility in agarose gels and sequence analysis indicate the amplicons are highly stable. The data presented establish this multiplex PCR as a reliable method for identification of B. bronchiseptica and both toxigenic and nontoxigenic P. multocida that may greatly simplify investigations of swine PAR and bronchopneumonia.
支气管败血波氏杆菌和多杀性巴氏杆菌是猪进行性萎缩性鼻炎（PAR）和支气管肺炎的病原体。只有产生皮肤坏死毒素的多杀性巴氏杆菌菌株在萎缩性鼻炎中起作用，而产毒菌株和无毒菌株都与肺炎有关。对涉及这些细菌的疫情进行监测和调查需要对多杀性巴氏杆菌分离株进行灵敏、准确的鉴定以及对其产毒状态进行可靠的判定。在本研究中，我们报告了一种多重PCR检测方法的开发、优化及性能特征，该方法可同时扩增多达三个不同的靶标，一个是所有多杀性巴氏杆菌菌株共有的，一个仅在产毒的多杀性巴氏杆菌菌株中发现，另一个是支气管败血波氏杆菌菌株共有的。基于对94株多杀性巴氏杆菌分离株（31株产毒）和126株支气管败血波氏杆菌分离株的分析，所有扩增子的检测灵敏度均为100%。对猪呼吸道中常见的其他22个细菌属和种的分离株进行评估，结果表明所有基因靶标的特异性均为100%。同时扩增所有靶标的检测限为每个靶标1 - 10 pg DNA，相当于几百个基因组或更少。扩增子在琼脂糖凝胶中的迁移率和序列分析表明扩增子高度稳定。所呈现的数据表明这种多重PCR是一种可靠的方法，可用于鉴定支气管败血波氏杆菌以及产毒和无毒的多杀性巴氏杆菌，这可能会大大简化对猪PAR和支气管肺炎的调查。
