Related Experiment Video
Updated: Jun 30, 2025
Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation
Published on: May 20, 2020
Development of a closed-tube, calcein-based loop-mediated isothermal amplification assay to detect Salmonella spp. in
Khristine B Balaga1, Rance Derrick N Pavon1, Alyzza Marie B Calayag1
1Pathogen-Host-Environment Interactions Research Laboratory, Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City 1101, Philippines.
Abstract:
Foodborne pathogens compromise food safety and public health, and Salmonella spp. are among the major pathogenic bacteria that cause outbreaks worldwide. Proper surveillance through timely and cost-effective detection methods across the food animal production chain is crucial to prevent Salmonella outbreaks and agricultural losses. Traditional culture methods are labor- and resource-intensive, with lengthy turnaround times. Meanwhile, conventional molecular tools, such as PCR and qPCR, are expensive and require technical skills and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, highly sensitive, and specific molecular assay that does not require expensive equipment. Hence, this study developed and optimized a closed-tube, calcein-based LAMP assay to detect Salmonella using the invA gene and performed evaluation and validation against conventional PCR. The LAMP assay showed high specificity and sensitivity. It showed 10-fold higher sensitivity than conventional PCR, at <1 ng/μL DNA concentrations. Meanwhile, for CFU/mL, LAMP assay showed 1000-fold higher sensitivity than conventional PCR at 4.8 × 103 cells/mL than 4.8 × 107 cells/mL, respectively. For parallel testing of 341 raw meat samples, after conventional culture enrichment (until Rappaport-Vassiliadis broth), the optimized LAMP assay showed 100% detection on all samples while conventional PCR showed 100%, 99.04%, and 96.64% for raw chicken, beef, and pork samples, respectively. Meanwhile, a shortened enrichment protocol involving 3-h incubation in buffered peptone water only, showed lower accuracy in tandem with the optimized LAMP assay ranging from 55 to 75% positivity rates among samples. These suggest that the optimized LAMP assay possesses higher sensitivity over conventional PCR for invA gene detection when coupled with conventional enrichment culture methods. Hence, this assay has potential as a powerful complementary or alternative Salmonella detection method to increase surveillance capacity and protect consumer food safety and public health worldwide.
