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Updated: Jun 29, 2025
A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.
Published on: November 7, 2018
Development and Application of Four Foodborne Pathogens by TaqMan Multiplex Real-Time PCR
Yinlei Xue1, Shengfang He2, Meng Li3
1Food Inspection and Testing Center of Yexian County, Yexian County, Henan, China.
Abstract:
A TaqMan multiplex real-time PCR (mRT-PCR) was developed to detect simultaneously Salmonella spp., Escherichia coli O157, Staphylococcus aureus, and Listeria monocytogenes in food samples. The method involves four sets of primers and probes tailored to the unique DNA sequences found in the invA, nuc, rfbE, and hly genes of each pathogen. The generated standard curves, correlating gene copy numbers with Ct values, demonstrated high accuracy (R2 > 0.99) and efficiency (92%-104%). Meanwhile, the limit of detection was 100 CFU/mL for the four target bacteria in artificially contaminated food samples after 6-8 h of enrichment. The assay's effectiveness was further verified by testing 80 naturally contaminated food samples, showing results largely in agreement with traditional culture methods. Overall, this newly developed TaqMan mRT-PCR, inclusive of a pre-enrichment step, proves to be a dependable and effective tool for detecting single or multiple pathogens in diverse food items, offering significant potential for in vitro diagnostics.
Insights
A new TaqMan multiplex real-time PCR (mRT-PCR) method accurately detects multiple foodborne pathogens, including Salmonella and E. coli O157, in food samples. This rapid assay offers a dependable solution for pathogen detection in food safety diagnostics.
Area of Science:
- Food Microbiology
- Molecular Diagnostics
- Pathogen Detection
Background:
- Foodborne pathogens pose significant public health risks.
- Accurate and rapid detection methods are crucial for food safety.
- Multiplex assays can improve efficiency in pathogen screening.
Purpose of the Study:
- To develop and validate a TaqMan multiplex real-time PCR (mRT-PCR) assay.
- To enable simultaneous detection of key foodborne pathogens.
- To provide a reliable tool for food safety testing.
Main Methods:
- Developed a mRT-PCR assay targeting invA, nuc, rfbE, and hly genes.
- Utilized specific primers and probes for Salmonella spp., E. coli O157, S. aureus, and L. monocytogenes.
- Validated the assay using artificially and naturally contaminated food samples.
Main Results:
- Achieved high accuracy (R² > 0.99) and efficiency (92%-104%) with standard curves.
- Established a limit of detection of 10⁰ CFU/mL after enrichment.
- Demonstrated good agreement with traditional culture methods in naturally contaminated samples.
Conclusions:
- The developed TaqMan mRT-PCR is a dependable and effective method.
- The assay allows for simultaneous detection of multiple pathogens in food.
- This tool has significant potential for in vitro diagnostics in food safety.
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