Real-time quantitative PCR for detection and identification of Actinobacillus pleuropneumoniae serotype 2

Abstract

Introduction: Porcine pleuropneumonia inflicts important economic losses on most commercial herds. Detection of subclinical or chronic infection in animals still remains a challenge, as isolation and identification of A. pleuropneumoniae serotypes is difficult and quantification of the bacteria on agar plates is often almost impossible. The aim of the study was to develop and evaluate a serotype-specific quantitative TaqMan probe-based PCR for detection of serotype 2 in pig lungs, tonsils, and nasal swabs. Material and Methods: The primers were designed from the capsular polysaccharide biosynthesis genes of A. pleuropneumoniae serotype 2. PCR specificity and sensitivity were evaluated using reference strains and several other bacterial species commonly isolated from pigs. Results: The real-time qPCR for detection of A. pleuropneumoniae serotype 2 was highly specific and gave no false positives with other serotypes or different bacterial species of pig origin. The detection limit for pure culture was 1.2 × 10 4 CFU/mL, for lung tissue and nasal swabs it was 1.2 × 10 5 CFU/mL, and for tonsils -1.2 × 10 5 CFU/mL. Conclusion: The method can be used to serotype A. pleuropneumoniae isolates obtained during cultivation and to detect and identify A. pleuropneumoniae serotype 2 directly in nasal swabs and tonsil scrapings obtained from live pigs or lung tissue and tonsils collected post-mortem.

Rapid Detection and Typing of Actinobacillus pleuropneumoniae Serovars Directly From Clinical Samples: Combining FTA® Card Technology With Multiplex PCR

“…However, for use in qPCR, it would be necessary to extract the DNA from FTA cards, rather than use a punched disc directly, as we have done with our mPCR assays. Furthermore, to date, diagnostic qPCRs for A. pleuropneumoniae have only been described for detection of the species-specific apxIV gene ( 35 ) and serotyping of a limited number of serovars ( 36 , 37 ). Further work will be required to extend diagnostic qPCRs to allow comprehensive serotyping of A. pleuropneumoniae compatible with the use of FTA card samples.…”