Listeria monocytogenes is a significant foodborne pathogen that can cause severe human listeriosis through contaminated milk. To address the need for rapid, sensitive, and specific detection, three methods were established in this study for detecting L. monocytogenes in milk: real-time quantitative PCR (qPCR), visual loop-mediated isothermal amplification (LAMP), and colloidal gold immunochromatographic test (GICT). A TaqMan probe targeting the lmo0733 gene was designed, and the optimized qPCR assay achieved a detection limit of 1.39×10 4 copies/μL for pure cultures and 3.21×10 3 CFU/mL in artificially contaminated milk. The visual LAMP method targeting the gyrB gene was completed within 20 min at 65°C, with results interpreted by color change. The detection limits were 1.135×10 3 copies/μL for pure cultures and 2.57×10 4 CFU/mL in artificially contaminated milk. In addition, a monoclonal antibody (titer 1:192,000) was prepared, and after optimization of labeling conditions, GICT was developed, enabling detection of L. monocytogenes at 1×10 5 CFU/mL within 10 min, with no cross-reactivity against other common milk-borne bacteria. All three methods exhibited high specificity and sensitivity, providing reliable tools for laboratory confirmation, on-site visual screening, and point-of-care testing, respectively. • Three rapid methods (qPCR, LAMP, GICT) were developed for L. monocytogenes detection in milk. • qPCR achieved a detection limit of 3.21×10 3 CFU/mL with high specificity. • Visual LAMP enabled detection within 20 min at 65°C with naked-eye interpretation. • GICT enabled instrument-free detection at 1×10 5 CFU/mL within 10-15 min. • All methods were validated using artificially contaminated and clinical milk samples.
Ma et al. (Fri,) studied this question.