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Simultaneous Detection of Four Foodborne Pathogens in Raw Freshwater Fish Using High-Resolution Melting Analysis
Shan Shan1,2, Xiaoyu Tong1, Wenyu Du1
1Nanchang Key Laboratory of Microbial Resources Exploitation & Utilization from Poyang Lake Wetland, College of Life Science, Jiangxi Normal University, Nanchang 330022, China.
Foods (Basel, Switzerland)
|September 27, 2025
Summary
A new multiplex PCR-HRM assay simultaneously detects four key foodborne pathogens in raw fish, enhancing food safety. This method offers a simple, efficient, and cost-effective solution for pathogen detection in aquatic products.
Area of Science:
- Food Science
- Microbiology
- Molecular Biology
Background:
- Raw fish consumption is popular globally but poses risks due to potential contamination with foodborne pathogens.
- Key pathogens like Listeria monocytogenes, Salmonella, Vibrio parahaemolyticus, and Staphylococcus aureus are common threats in raw seafood.
- Accurate and rapid detection methods are crucial for ensuring the safety of aquatic products.
Purpose of the Study:
- To develop and validate a novel multiplex assay for the simultaneous detection of four major foodborne pathogens in raw fish.
- To establish a high-resolution melting (HRM) curve analysis integrated with multiplex PCR for pathogen identification.
- To assess the sensitivity, specificity, and efficiency of the developed assay for food safety applications.
Main Methods:
- Multiplex polymerase chain reaction (PCR) was employed to amplify target genes from four specific foodborne pathogens.
- High-resolution melting (HRM) curve analysis was utilized to differentiate the amplified products based on melting temperature (Tm).
- The assay's performance was evaluated using various concentrations of genomic DNA, specificity testing with non-target strains, and sensitivity analysis.
Main Results:
- The PCR-HRM assay successfully detected Listeria monocytogenes, Salmonella, Vibrio parahaemolyticus, and Staphylococcus aureus simultaneously.
- The detection limit ranged from 0.02-0.1 ng/µL, demonstrating high sensitivity.
- The assay exhibited excellent specificity and accuracy, with no interference from co-extracted DNA of non-target bacteria.
Conclusions:
- The integrated multiplex PCR-HRM assay is a highly efficient, accurate, and cost-effective method for detecting multiple foodborne pathogens in raw fish.
- This assay significantly reduces detection time and operational complexity, making it ideal for routine food safety monitoring.
- The developed method provides a robust tool for enhancing safety standards in the aquatic products processing industry.
