Evaluation of the performance of a point-of-care molecular assay for simultaneous detection of the agents of American foulbrood and European foulbrood in apiaries

American foulbrood (AFB) and European foulbrood (EFB), caused by <i>Paenibacillus larvae</i> and <i>Melissococcus plutonius</i>, respectively, are severe bacterial diseases that significantly affect honey bee health and productivity worldwide. Rapid and accurate diagnosis is essential for effective disease management in apiaries. We developed and validated a multiplex point-of-care (POC) quantitative real-time PCR (qPCR) assay that enables simultaneous and rapid detection of <i>P. larvae</i> and <i>M. plutonius</i> directly in apiaries. Our POC qPCR assay, using the XQ station (Postbio), had diagnostic sensitivity and specificity comparable to laboratory-based qPCR assays. For <i>P. larvae</i> detection, the POC qPCR assay had a sensitivity of 93% (56 of 60 samples) and specificity of 97% (97 of 100 samples). Similarly, for <i>M. plutonius</i>, sensitivity was 95% (59 of 62 samples) and specificity was 97% (97 of 100 samples). Our POC qPCR assay had analytical sensitivity comparable to that of laboratory-based qPCR assays, with a limit of detection of ~10² copies/reaction for both pathogens. Additionally, the assay had high analytical specificity, with no cross-reactivity against other common honey bee pathogens, including deformed wing virus (DWV), black queen cell virus (BQCV), and <i>Vairimorpha ceranae</i>. Our POC qPCR assay consistently had lower cycle quantification values than the laboratory-based qPCR on the same samples, indicating robust detection capability even at low pathogen concentrations. Although not compared to a gold standard, our POC qPCR assay had reliable diagnostic performance. Our multiplex POC qPCR assay enables rapid, accurate pathogen detection in apiaries, thereby enhancing disease management and supporting sustainable, productive apiculture.