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Updated: Oct 5, 2025
Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation
Published on: May 20, 2020
Mpl-Gene-Based Loop-Mediated Isothermal Amplification Assay for Specific and Rapid Detection of Listeria
Annemarie Busch1, André Becker1, Ulrich Schotte2
1Institute of Food Quality and Food Safety, University of Veterinary Medicine Hannover, Hannover, Germany.
Abstract:
Listeria monocytogenes represents a high risk in food and can trigger potentially fatal listeriosis. The objective of this study was to detect L. monocytogenes in food using the LAMP method in a fast, specific, sensitive manner and thus to preventively test food for the presence of the target species. The reaction was performed and established using the portable real-time fluorometer Genie® II (OptiGene Ltd., Horsham, United Kingdom). In this new assay, six LAMP primers targeted the mpl-gene sequence of L. monocytogenes. A total of 148 different isolates, including 105 L. monocytogenes and 43 non-L. monocytogenes strains, were tested. Analytical sensitivity was determined based on different DNA- and cell concentrations. The detection limit with a detection rate of 100% was 5 pg of DNA or 275 colony-forming units (CFU) per reaction. Artificially contaminated minced beef and grated mozzarella were also tested. The assay was 100% successful to detect an initial bacterial contamination of 0.4-4 CFU g-1 food after 24 h enrichment in half-Fraser broth. Finally, natively contaminated samples were tested in comparison to the microbiological reference method and real-time polymerase chain reaction. Native sample testing revealed 100% consistent findings between LAMP and the standard culture method after first enrichment for 24 h. In addition, a rapid colony-confirmation method was established that enabled reliable identification of L. monocytogenes isolates on different selective culture media using a simplified DNA extraction by boiling. This study showed that the developed assay was able to determine whether a food is safe with respect to the food-safety criteria of 100 CFU per gram, according to standards of the European Union, for L. monocytogenes and provided faster results than the cultural reference method.
Insights
A new Loop-mediated Isothermal Amplification (LAMP) assay quickly and accurately detects Listeria monocytogenes in food. This method ensures food safety by identifying the pathogen faster than traditional methods.
Area of Science:
- Food Microbiology
- Molecular Diagnostics
- Bacterial Pathogen Detection
Background:
- Listeria monocytogenes poses a significant food safety risk, potentially causing fatal listeriosis.
- Accurate and rapid detection methods are crucial for preventing foodborne illness outbreaks.
- Existing methods can be time-consuming, delaying food safety assessments.
Purpose of the Study:
- To develop a fast, specific, and sensitive Loop-mediated Isothermal Amplification (LAMP) assay for detecting Listeria monocytogenes in food.
- To enable rapid, preventive testing of food products for Listeria monocytogenes contamination.
- To establish a simplified DNA extraction method for rapid colony confirmation.
Main Methods:
- A novel LAMP assay was designed using six primers targeting the mpl-gene of Listeria monocytogenes.
- The assay was optimized and validated using a portable real-time fluorometer.
- Analytical sensitivity was assessed using various DNA and cell concentrations, and artificial/native food samples were tested.
Main Results:
- The LAMP assay demonstrated high analytical sensitivity with a detection limit of 5 pg DNA or 275 CFU/reaction.
- It successfully detected low levels of Listeria monocytogenes (0.4-4 CFU/g) in artificially contaminated beef and mozzarella after enrichment.
- Testing of native samples showed 100% agreement with standard culture methods after 24-hour enrichment.
Conclusions:
- The developed LAMP assay provides a rapid, sensitive, and specific method for Listeria monocytogenes detection in food.
- This assay meets European Union food safety criteria (100 CFU/g) and offers faster results than conventional methods.
- A simplified DNA extraction method allows for rapid confirmation of Listeria monocytogenes colonies.
