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Updated: May 8, 2026
A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.
Published on: November 7, 2018
Multiplex TaqMan® detection of pathogenic and multi-drug resistant Salmonella
Prashant Singh1, Azlin Mustapha
1Food Science Program, University of Missouri, Columbia, MO 65211, USA.
Abstract:
Overuse of antibiotics in the medical and animal industries is one of the major causes for the development of multi-drug-resistant (MDR) food pathogens that are often difficult to treat. In the past few years, higher incidences of outbreaks caused by MDR Salmonella have been increasingly documented. The objective of this study was to develop a rapid multiplex real-time polymerase chain reaction (PCR) assay for simultaneous detection of pathogenic and MDR Salmonella spp. A multiplex TaqMan®real-time PCR was designed by targeting the invasin virulence gene (invA), and four commonly found antibiotic resistance genes, viz. ampicillin, chloramphenicol, streptomycin and tetracycline. To avoid false negative results and to increase the reliability of the assay, an internal amplification control (IAC) was added which was detected using a locked nucleic acid (LNA) probe. In serially diluted (5 ng-50 fg) DNA samples, the assay was able to detect 100 genomic equivalents of Salmonella, while in a multiplex format, the sensitivity was 1000 genomic equivalents. The assay performed equally well on artificially contaminated samples of beef trim, ground beef of different fat contents (73:27, 80:20, 85:15 and 93:7), chicken rinse, ground chicken, ground turkey, egg, spinach and tomato. While the detection limit for un-enriched inoculated food samples was 10(4) CFU/g, this was improved to 10 CFU/g after a 12-h enrichment in buffered peptone water, with 100% reproducibility. The multiplex real-time assay developed in this study can be used as a valuable tool to detect MDR virulent Salmonella, thus enhancing the safety of food.
Insights
This study developed a rapid multiplex real-time polymerase chain reaction (PCR) assay to detect multi-drug-resistant (MDR) Salmonella. The assay enhances food safety by identifying virulent Salmonella strains and their antibiotic resistance genes simultaneously.
Area of Science:
- Food Safety
- Microbiology
- Molecular Biology
Background:
- Antibiotic overuse drives multi-drug-resistant (MDR) foodborne pathogens, with increasing Salmonella outbreaks.
- Rapid detection of MDR Salmonella is crucial for public health and food safety.
Purpose of the Study:
- To develop a rapid multiplex real-time polymerase chain reaction (PCR) assay for simultaneous detection of pathogenic and MDR Salmonella spp.
- To target key virulence and antibiotic resistance genes for comprehensive Salmonella identification.
Main Methods:
- Designed a multiplex TaqMan® real-time PCR assay targeting the invA gene and four antibiotic resistance genes (ampicillin, chloramphenicol, streptomycin, tetracycline).
- Incorporated an internal amplification control (IAC) with a locked nucleic acid (LNA) probe to ensure assay reliability.
- Validated the assay on various food matrices (beef, chicken, turkey, egg, spinach, tomato) and assessed sensitivity and reproducibility.
Main Results:
- The assay detected 100 genomic equivalents of Salmonella in singleplex and 1000 genomic equivalents in multiplex format.
- Achieved a detection limit of 10 CFU/g in food samples after 12-h enrichment, with 100% reproducibility.
- Demonstrated effective performance across diverse food types, including those with varying fat content.
Conclusions:
- The developed multiplex real-time PCR assay is a valuable tool for rapid and simultaneous detection of MDR virulent Salmonella.
- This assay can significantly enhance food safety by enabling timely identification of contamination.
- The method offers a reliable approach for monitoring and controlling MDR Salmonella in the food supply chain.
