Work

Development of recombinase-aided amplification assays with real-time fluorescence and lateral flow dipstick for the rapid detection of Actinobacillus pleuropneumoniae

Year: 2025

Type: article

Abstract: 1 The recombinase-aided amplification (RAA) assays were developed for the rapid detection of A. pleuropneumoniae. 2 The results can not only be detected by real-time fluorescence readout but also can be visualized by a portable blue light instrument and lateral flow dipsticks. 3 These assays provided reliable tools for preventing A. pleuropneumonia , especially in on-site diagnostics. Porcine Contagious Pleuropneumonia (PCP) is an important bacterial infectious disease caused by Actinobacillus pleuropneumoniae ( A. pleuropneumoniae ), which has caused serious economic losses to the pig industry. Early rapid diagnosis and pathogen surveillance are essential for the prevention of this disease. In this study, recombinase-aided amplification (RAA) assays with real-time fluorescence and lateral flow dipsticks (LFD) for the rapid detection of A. pleuropneumoniae were established, optimized, and evaluated based on the conserved region of the apxIVA gene. The results of the exo-probe-based RAA assay were not only determined by real-time fluorescence readout (real-time RAA) but also by a portable blue light instrument for visualization (visual RAA). The results of the nfo-probe-based RAA assay were determined by LFDs (RAA-LFD). These assays could detect all serotypes of A. pleuropneumoniae and had no cross-reaction with other common clinical pathogenic bacteria. The analytical sensitivity of the real-time RAA and RAA-LFD assays was 17.9 copies µL -1 with a 95% confidence interval, and the analytical sensitivity of the visual RAA assay was 203.5 copies µL -1 with a 95% confidence interval. The coincidence rate of real-time RAA assay with the qPCR assay was 97.6%, and the coincidence rate of visual RAA and RAA-LFD assays with the qPCR assay was 100% in detecting clinical samples. The RAA assays have high specificity, high sensitivity, and good diagnostic performance, which can be used as reliable diagnostic tools for the rapid detection of A. pleuropneumoniae , especially in on-site diagnostics.

Source: Journal of Integrative Agriculture

DOI: 10.1016/j.jia.2025.05.023