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Published on: June 9, 2014
Establishment of a high-throughput xMAP technology for detection of 16 pathogens in raw milk
Na Liu1,2,3, Sha Xiao1,2,3, Jianmei Zhao1,2,3
1China Animal Health and Epidemiology Center, Qingdao 266032, China.
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.
Insights
A new multiplex assay rapidly detects 16 raw milk pathogens using fluorescent microspheres. This method offers high specificity and sensitivity for routine pathogen monitoring in raw milk.
Area of Science:
- Food safety
- Microbiology
- Biotechnology
Background:
- Raw milk can harbor various pathogens, posing risks to public health.
- Accurate and rapid detection methods are crucial for effective food safety monitoring.
- Existing methods may lack the speed or multiplexing capability for comprehensive screening.
Purpose of the Study:
- To develop and validate a rapid, simultaneous detection method for 16 common raw milk pathogens.
- To optimize reaction parameters for a novel multiplex assay.
- To assess the assay's specificity, sensitivity, reproducibility, and applicability in real-world samples.
Main Methods:
- Design and modification of species-specific primers and probes for 16 target pathogens.
- Coupling of probes with fluorescent carboxylated microspheres for multiplexing.
- Optimization of critical reaction parameters, including hybridization temperature and duration.
- Validation using positive controls, analytical sensitivity testing, reproducibility assessment, and analysis of artificially contaminated raw milk samples.
Main Results:
- The developed xMAP-based assay achieved simultaneous detection of 16 pathogens with distinct fluorescence signals and no cross-hybridization.
- Analytical sensitivity reached 10 CFU mL-1 for eight pathogens and 102 CFU mL-1 for the other eight.
- High reproducibility was confirmed (coefficients of variation 6.23%–13.4%).
- Validation with artificially contaminated raw milk showed comparable results to PCR and culture-based methods.
Conclusions:
- The established xMAP multiplex detection system provides a highly specific, sensitive, and high-throughput solution for raw milk pathogen monitoring.
- This assay is suitable for routine application in ensuring the safety of raw milk.
- The method offers a significant advancement in rapid pathogen detection for the dairy industry.
