Development of Recombinase Polymerase Amplification (RPA) combined SYBR Green I Dye Assay for Rapid Detection of Lipase-Producing Psychrotrophic Bacteria in Raw Milk

• The assay was time-saving, sensitive, easy to operate, no need for laboratory. • The LOD of RPA was 100-fold sensitive than conventional PCR, dropped to 10 2 CFU/mL. • Achieve on-site detection of bacteria in 1 hour. Rapid detection of psychrotrophic bacteria from raw milk is essential for diary product safety and quality, as these organisms proliferate during cold-chain transport and secrete heat-resistant lipase remaining active after UHT (Ultra-High Temperature) processing, subsequently leading to off-flavors and spoilage. In this study, a recombinase polymerase amplification (RPA) assay targeting a conserved segment of the triacylglycerol lipase gene ( lipA ) was established for 8 psychrotrophic bacteria, including a novel strain of Pseudomonas fluorescens ( P. fluorescens ) strain frequently reported psychrotrophic bacteria isolated from raw milk. The RPA primers were designed based on a conserved region of lipA sequence through multi-sequence alignment. The RPA reaction conditions were optimized, including primer, MgOAc, temperature, time. The RPA limit of detection (LOD) for pure culture was 10 pg/µL, 100-fold more sensitive than conventional PCR method. The integration of a colorimetric readout further enhanced the assay's sensitivity, reducing the LOD in artificially contaminated UHT milk to 1.2 × 10° CFU/mL—representing an additional 100‑fold improvement, and the whole detection could be finished in 1 hour including 45 min for the rapid DNA extraction and 15 min for RPA detection. This platform demonstrated strong suitability for on‑site deployment. Particularly when integrated with CRISPR-Cas12 or lateral-flow dipsticks, it could provide an effective alternative for monitoring psychrotrophic spoilage bacteria in chilled raw milk.