Direct detection of Actinobacillus pleuropneumoniae in swine lungs and tonsils by real-time recombinase polymerase amplification assay. (June 2019)

• Record Type: Journal Article
• Authors: Li, Ruiwen; Wang, Jinfeng; Liu, Libing; Zhang, Ruoxi; Hao, Xuepiao; Han, Qingan; Wang, Jianchang; Yuan, Wanzhe
• Is Part Of: Molecular and cellular probes. Volume 45(2019)
• Page Start: 14
• Page End: 18
• Publication Date: 2019-06
• Subjects: Actinobacillus pleuropneumoniae -- apxIVA gene -- Real-time RPA -- Isothermal amplification; Molecular probes -- Diagnostic use -- Periodicals; Pathology, Cellular -- Technique -- Periodicals; Cell Biology -- Periodicals; Molecular Biology -- Periodicals; Sondes moléculaires -- Utilisation diagnostique -- Périodiques; Cytopathologie -- Technique -- Périodiques; 572
• DOI: 10.1016/j.mcp.2019.03.007
• Languages: English
• ISSNs: 0890-8508

Abstract

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. Highlights: A real-time RPA assay was developed for rapid, specific and sensitive detection of A. pleuropneumoniae. Assay was performed successfully at a constant temperature of 39 °C within 20 min. Clinical swine lungs and tonsils were used for further assay evaluation. Assay was demonstrated to be simple, rapid and reliable in A. pleuropneumoniae detection.