Lo T M, Ward C K, Inzana T J
Center for Molecular Medicine and Infectious Disease, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg 24061-0342, USA.
J Clin Microbiol. 1998 Jun;36(6):1704-10. doi: 10.1128/JCM.36.6.1704-1710.1998.
Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10(2) CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.
胸膜肺炎放线杆菌的血清分型基于血清型特异性荚膜抗原的检测。然而，并非所有分离株都能进行血清分型，有些可能会与多种血清分型试剂发生交叉反应。为了提高血清分型的灵敏度和特异性以及实现早期检测，开发了一种多重聚合酶链反应（PCR）检测方法，用于检测胸膜肺炎放线杆菌并鉴定血清型5分离株。胸膜肺炎放线杆菌血清型5荚膜多糖保守输出区域和血清型特异性生物合成区域的特异性DNA序列分别用作引物，以扩增0.7 kb和1.1 kb的DNA片段。除血清型4外，在所检测的所有胸膜肺炎放线杆菌菌株中均扩增出0.7 kb的片段。未从猪的常见病原体或共生菌等任何异源物种中扩增出0.7 kb的片段。相比之下，仅从所有血清型5菌株中扩增出1.1 kb的片段。该检测方法能够从少于10²CFU中扩增出DNA。尽管从一些冷冻保存6年的组织中未扩增出1.1 kb的产物，但胸膜肺炎放线杆菌血清型5荚膜DNA产物很容易从感染猪的肺组织中扩增出来。多重PCR检测方法使我们能够快速检测胸膜肺炎放线杆菌，并将血清型5菌株与其他血清型区分开来。使用针对其他胸膜肺炎放线杆菌血清型生物合成区域的引物将扩展该检测方法的诊断和流行病学能力。
