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Updated: Jul 11, 2025
Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation
Published on: May 20, 2020
Novel helix loop-mediated isothermal amplification (HAMP) assay for colorimetric detection of Staphylococcus aureus
A Arun Prince Milton1, M C B Prasad2, G Bhuvana Priya3
1Division of Animal and Fisheries Sciences, ICAR Research Complex for NEH Region, Umiam, Meghalaya, India. vetmilton@gmail.com.
Abstract:
Staphylococcus aureus is an important and leading cause of foodborne diseases worldwide. Prompt detection and recall of contaminated foods are crucial to prevent untoward health consequences caused by S. aureus. Helix loop-mediated isothermal amplification (HAMP) is an exciting recent addition to the array of available isothermal-based nucleic acid amplification techniques. This study aimed to develop and evaluate a HAMP assay for detecting S. aureus in milk and milk products. The assay is completed in 75 minutes of isothermal temperature incubation (64 ˚C) and dye-based visual interpretation of results based on colour change. The specificity of the developed assay was ascertained using 27 S. aureus and 17 non S. aureus bacterial strains. The analytical sensitivity of the developed HAMP assay was 9.7 fg/µL of pure S. aureus DNA. The detection limit of the HAMP assay in milk (86 CFU/mL) was 1000x greater than the routinely used endpoint PCR (86 × 103 CFU/mL). The practicality of applying the HAMP assay was also assessed by analysing milk and milk product samples (n = 95) obtained from different dairy farms and retail outlets. The developed test is a more rapid, sensitive, and user-friendly method for the high-throughput screening of S. aureus in food samples and may therefore be suitable for field laboratories. To our knowledge, this is the first study to develop and evaluate the HAMP platform for detecting S. aureus.
Insights
A new Helix loop-mediated isothermal amplification (HAMP) assay rapidly detects Staphylococcus aureus in milk. This sensitive and user-friendly method offers high-throughput screening for food safety.
Area of Science:
- Food Microbiology
- Molecular Diagnostics
- Isothermal Amplification Techniques
Background:
- Staphylococcus aureus is a primary cause of global foodborne illness.
- Rapid detection and recall of contaminated foods are essential for public health.
- Isothermal nucleic acid amplification techniques offer alternatives to traditional PCR.
Purpose of the Study:
- To develop and validate a Helix loop-mediated isothermal amplification (HAMP) assay for Staphylococcus aureus detection.
- To evaluate the assay's performance in milk and milk products.
- To assess the assay's suitability for field laboratory applications.
Main Methods:
- Development of a HAMP assay with a 75-minute incubation at 64°C.
- Visual result interpretation using a dye-based colorimetric change.
- Specificity testing with 27 S. aureus and 17 non-S. aureus strains.
- Analytical sensitivity determined at 9.7 fg/µL DNA.
- Detection limit in milk assessed and compared to endpoint PCR.
- Practicality evaluated using 95 milk and milk product samples.
Main Results:
- The HAMP assay demonstrated high specificity.
- Analytical sensitivity was 9.7 fg/µL of S. aureus DNA.
- The assay's detection limit in milk (86 CFU/mL) was 1000 times better than endpoint PCR.
- Practicality assessment confirmed its applicability to real-world samples.
- The assay provides rapid, sensitive, and user-friendly detection.
Conclusions:
- The developed HAMP assay is a rapid, sensitive, and user-friendly method for high-throughput screening of S. aureus in food samples.
- This assay is suitable for use in field laboratories.
- This represents the first study to develop and evaluate the HAMP platform for S. aureus detection.
