Establishment of a high-throughput xMAP technology for detection of 16 pathogens in raw milk.

Abstract

To establish a rapid detection method for pathogens potentially contaminating raw milk, species-specific primers and probes were designed and modified for the simultaneous identification of 16 different target pathogens. Probes were coupled with fluorescent carboxylated microspheres, while specific positive plasmids and strains constructed previously were positive controls. Critical reaction parameters, including hybridization temperature (52°C) and duration (20 min), were systematically optimized. The assay demonstrated excellent specificity, with each probe exhibiting distinct fluorescence signals and no observable cross-hybridization. Analytical sensitivity test revealed detection limits of 10 CFU mL-1 for eight pathogens, and 102 CFU mL-1 for the remaining eight. Reproducibility was confirmed, with coefficients of variation ranging from 6.23% to 13.4%. Validation using artificially contaminated raw milk samples (six pathogens) demonstrated similar results between the xMAP assay and two reference methods (PCR and culture-based). The established xMAP-based multiplex detection system exhibits high specificity, sensitivity, and throughput, demonstrating its applicability for routine monitoring of raw milk pathogens.