Development of a Rapid RAA-CRISPR-Based Method for Detecting Single-Nucleotide Polymorphisms in Pseudomonas lundensis

Abstract Pseudomonas species are prevalent psychrotrophic spoilage bacteria in dairy products, and the heat-stable protease AprX secreted by these bacteria is a major contributor to milk quality deterioration. Different strains exhibit significant variation in spoilage potential, underscoring the necessity for strain-level detection methods that can identify highly spoilage-prone subtypes. In this study, we developed an RAA-CRISPR/Cas12a-based detection method targeting a single-nucleotide polymorphism (SNP, position 336 G→A) in the aprX gene of Pseudomonas lundensis. This SNP enables specific discrimination of the highly spoilage-prone P. lundensis strains identified in our previous research. Orthogonal optimization identified optimal RAA conditions (2 μL template, 1.2 μL primers, 39 °C), and we determined that a CRISPR/Cas12a reaction temperature of 42 °C effectively discriminated between highly spoilage-prone and weakly spoilage-prone strains, with the latter producing no detectable fluorescence. The method demonstrated a detection limit of 103 copies/μL for the recombinant plasmid, with a quantifiable range of 103–107 copies/μL. In artificially contaminated milk, following large-volume centrifugal enrichment, the detection limit was achieved at 102 CFU/mL, with a quantifiable range of 102–108 CFU/mL (R2 > 0.99). This study represents the first precise identification of a specific SNP site in P. lundensis for distinguishing highly spoilage-prone strains, providing a rapid, sensitive, and specific novel tool for early warning of high-risk spoilage bacteria in dairy products.