A rapid and specific loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for detecting Melissococcus plutonius, the causal agent of European foulbrood in honeybees.

Melissococcus plutonius, a gram-positive bacterium, causes European Foulbrood (EFB) disease in honeybee larvae, leading to reduced yields in various countries. This study developed a rapid detection method for M. plutonius using colorimetric loop-mediated isothermal amplification with a lateral flow dipstick (cLAMP-LFD), based on the sequences of gyrase subunit B (gyrB) and superoxide dismutase (sodA) genes, giving three primer sets (gyrB1, gyrB2, and sodA). The cLAMP-LFD using gyrB1 was capable of detecting M. plutonius specifically and sensitively under the optimal condition of 63°C for 50 min with no cross-reactivity with other bacteria, and the limit of detection (LOD) was 2 × 104 copies per reaction and 50 CFU of bacterial cells per reaction. In spiked samples, the assay successfully detected M. plutonius in honeybee larvae and adult honeybees, with minimum detectable levels of 11.4 and 80 CFU per reaction, respectively. Our results demonstrate that the gyrB based 1cLAMP-LFD assay is suitable for detecting M. plutonius in apiary settings and in commercial honeybee samples, including those that tested negative by conventional methods. Overall, this cLAMP-LFD assay represents a rapid, sensitive, and specific tool for M. plutonius detection and may facilitate field surveillance, disease control, and management of EFB outbreaks worldwide, thereby supporting improved honey production efficiency.