High-throughput nucleic acid extraction using a pipette-tip annular magnetic separator integrated with real-time fluorescent RPA for rapid detection of Burkholderia gladioli pv. cocovenenans

Burkholderia gladioli pv . cocovenenans (BGC) is an emerging foodborne pathogen capable of producing the deadly toxins bongkrekic acid and toxoflavin, posing significant risks to public health. Currently, rapid screening for this bacterium is severely challenged by the inherently complex nature of various food matrices and the low throughput of routine nucleic acid extraction. Here we report a pipette-tip-compatible annular magnetic separator (AMS) that efficiently operates with magnetic silica beads in 96-well plate format. This innovative device enables parallel DNA extraction of 96 samples within merely 13.5 min without heating or centrifugation, thus streamlining the pretreatment process. This platform integrates the high-throughput extraction step with real-time fluorescent recombinase polymerase amplification (RT-RPA) specifically targeting the Bon M gene for the rapid detection of BGC in food products. Under optimized conditions, the integrated AMS-RT-RPA platform provides extraction-to-detection diagnostic results in 33.5 min (comprising 13.5 min for DNA extraction and approximately 20 min for RT-RPA amplification), and achieves a limit of detection as low as 190 CFU/mL. Furthermore, the spiked samples of black fungus and rice flour were also tested by the detection platform, and the recoveries ranged from 89.7% to 103.2%, with the coefficient of variation less than 11.6%. Collectively, this work demonstrates the practical feasibility of a scalable pretreatment-amplification workflow, highlighting its potential for high-throughput on-site screening during foodborne emergency investigations and routine food safety monitoring.