Thermostable alkaline protease (TAP) harbored by Pseudomonas fluorescens decomposes protein in milk and dairy products, leading to milk and dairy product spoilage during storage. Thus, a specific, sensitive, rapid, and simple method is required to detect TAP-harboring P. fluorescens. Two sets of primers targeting the aprX and gyrB genes of P. fluorescens were designed. The detection system and conditions were optimized, and a real-time loop mediated isothermal amplification (real-time LAMP) method was developed for the simultaneous detection of TAP-harboring P. fluorescens in two separate reaction tubes. The phylogenetic tree targeting aprX showed that P. fluorescens and P. lurida clustered on the same branch. The phylogenetic tree targeting gyrB showed that P. fluorescens clustered on the same branch with 95% confidence value, whereas P. lurida clustered on different branches. DNA of 16 strains of P. fluorescens and 34 strains of non-P. fluorescens was detected by real-time LAMP. TAP-harboring P. fluorescens can only be identified when the real-time LAMP detection results of both aprX and gyrB are positive. The dissociation temperatures of aprX and gyrB in the real-time LAMP amplified products were approximately 90.0 °C and 88.0 °C, respectively. The detection limits of the real-time LAMP targeting aprX and gyrB were 4.9 CFU/reaction in pure culture and 2.2 CFU/reaction in skimmed milk. The coefficient of variation of the repeatability test was less than 2%, indicating that the established real-time LAMP of P. fluorescens targeting gyrB and aprX has good stability and repeatability. Two-hundred raw milk samples were tested for the presence of TAP-harboring P. fluorescens by real-time LAMP in 3 h, and the coincidence rate of the results with those obtained using the traditional method, which takes at least 5-7 d, was 100%. Real-time LAMP will be a practical and effective method for accurate and rapid identification of TAP-harboring P. fluorescens in raw milk.

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