Identification and Detection of Actinobacillus pleuropneumoniae in Infected and Subclinically Infected Pigs by Multiplex PCR Based on the Genes ApxIVA and OmlA

PCRs based on different genes of Actinobacillus pleuropneumoniae have been developed for detecting and identifying A. pleuropneumoniae. Some of them could amplify positive fragments from the phylogenetically closely related species bacteria. To improve veracity and specificity of PCR, a species-specific multiplex PCR assay was developed to identify and detect A. pleuropneumoniae, based on the 3＇-terminus of the species-specific apxlVA gene and the already existing species-specific primers in the omlA gene. Both 346-bp and 950-bp fragments could be simultaneously amplified from all A. pleuropneumoniae reference strains and isolates, and the species specificity of the assay was evaluated with a collection of ten strains representing eight different species bacteria including species normally found in the respiratory tracts of swine. All of these strains turned out negative in the multiplex PCR. All sequences of products of multiplex PCR randomly sampled were also correct. The sensitivity of the multiplex PCR was determined to be 10 pg of A. pleuropneumoniae DNA. The multiplex PCR and bacterial isolation were compared to determine their sensitivities by using experimentally infected pigs and clinical disease pigs. The multiplex PCR was more sensitive than bacterial isolation. The multiplex PCR was also evaluated on mixed bacterial cultures from clinical healthy pigs. 26/100 （26%） of the subclinically infected pigs were detected from clinical healthy pigs. The results indicate that the multiplex PCR assay is a sensitive, highly specific, and effective diagnostic tool for identification and detection of A. pleuropneumoniae.

猪胸膜肺炎放线杆菌江西分离株ApxIVA基因PCR扩增及基因分析

猪胸膜肺炎是引起保育到育肥阶段猪生产性能下降的一种重要传染病,该病的发生往往与猪瘟、猪繁殖与呼吸综合征等病毒混合感染,引起猪群大批死亡,严重危害养猪生产。该病的病原为胸膜肺炎放线菌(APP),为促进江西省养猪业的发展,控制猪胸膜肺炎的流行,项目组从病原入手进行研究,前期已经分离到多株APP,并已经初步鉴定血清型。试验针对其毒素ApxIVA基因进行研究,参考NCBI上登录号为AX002405的ApxIVA毒素基因,设计目的基因大小为1 284 bp的特异性引物1对,并在上下游引物内加入酶切位点EcoRⅠ与SalⅠ进行毒素基因克隆。结果表明江西源APP应用PCR方法扩增ApxIVA基因,产物插入克隆载体,测序后与NCBI发表的APP ApxIVA基因序列对比分析,同源在98%以上,表明成功克隆到APP的ApxIVA基因。通过与10个国内外APP基因序列对比,发现本菌株与CP000569、FJ848575、FJ848573、GQ332268同源性较高,其中FJ848575、FJ848573、GQ332268均为中国分离株,CP000569为加拿大分离株。

猪传染性胸膜肺炎放线杆菌LAMP方法的建立

为建立快速检测猪传染性胸膜肺炎放线杆菌（APP）的鉴别方法，衣研究根据APP ApxIVA基因的保守区设计6条环介导等温扩增（LAMP）引物，利用Loopamp Realtime Turbidimeter LA-320c实时浊度仪对反应体系和条件进行优化，建立了APP的LAMP方法。结果显示：该方法在64℃下反应15rainDNA即可出现扩增，扩增后2～3min内即可判定结果，最低检测限为0．307ng／L，具有良好的重复性及稳定性，与其他病原菌无交叉反应，同时，试验结果可实现肉眼可视化观察。采用建立的LAMP方法与SN／T14472011标准公布的方法同时对36份临床疑似APP样品进行检测分析，结果显示：APP阳性10份，阴性26份，两种方法的符合率为100％。本研究建立的方法为防止猪传染性胸膜肺炎的传播提供了有效的检测手段。