Direct detection of Actinobacillus pleuropneumoniae in swine lungs and tonsils by real-time recombinase polymerase amplification assay

Abstract

Actinobacillus pleuropneumoniae is the etiological agent of swine contagious pleuropneumoniae, which is distributed globally and associated with severe economic losses in the pig rearing industry. In this study, a real-time recombinase polymerase amplification assay (real-time RPA) based on the apxIVA gene was developed to rapid detect A. pleuropneumoniae. Real-time RPA was performed successfully in Genie III at the constant temperature of 39 °C for 20 min. The developed assay was highly specific for A. pleuropneumoniae, and the sensitivity at 95% probability was 536 fg of A. pleuropneumoniae genomic DNA. The real-time RPA for A. pleuropneumoniae was further evaluated on the 112 clinical swine lung and tonsil samples, and 25 (22.3%), 27 (24.1%), and 12 (10.7%) samples were positive for A. pleuropneumoniae by the real-time RPA, real-time PCR and bacterial isolation, respectively. With a real-time PCR as the reference method, the real-time RPA showed a diagnostic specificity of 98.8%, a diagnostic sensitivity of 88.9%, a positive predicative value of 96.0%, a negative predictive value of 96.5%, and a kappa value of 0.900. The above data demonstrated the well potentiality and usefulness of the developed real-time RPA assay in the reliable detection of A. pleuropneumoniae, especially in resource limited settings.