by: Josiah Caleb Magollado | Chromoplexy
Salmonellosis, a global health challenge caused by the gram-positive bacterium Salmonella, primarily affects the intestines. Outbreaks have been particularly prevalent in the Philippines, where cases rose from 9,000 in 2022 to 13,000 in 2023. This illness often comes from contaminated foods like meat, eggs, dairy, and produce. Traditional detection relies on Polymerase Chain Reaction (PCR), a method that amplifies specific DNA regions through thermocycling—repeated cycles of heating and cooling—which trigger DNA synthesis. In Salmonella detection, PCR targets specific sequences in bacterial DNA to confirm its presence in a sample. Although highly sensitive, PCR’s open-tube design makes it prone to contamination, requires expensive equipment and skilled technicians, and can be time-consuming.
To address these limitations, Rance Derrick Pavon and Khristine Balaga, graduate students from the University of the Philippines Diliman - Institute of Biology (UPD IB), developed a more practical solution: a closed-tube, calcein-based Loop-Mediated Isothermal Amplification (LAMP) assay tailored for Salmonella enterica. Unlike PCR, LAMP amplifies DNA at a constant, lower temperature (60-65°C), using multiple primers and Bst Polymerase to increase specificity. The LAMP’s closed-tube design allows real-time visual detection through color change—green for positive, orange for negative—eliminating the need for complex post-amplification processing and reducing contamination risk.
In tests with 341 raw meat samples (i.e., chicken, beef, and pork), LAMP exhibited 10 times greater sensitivity than PCR in DNA detection and 1,000 times greater in bacterial cell detection. While a shortened culture enrichment protocol lowered LAMP’s accuracy, following conventional culture enrichment (i.e., longer incubation time) enabled LAMP to detect Salmonella with 100% accuracy across all samples, outperforming PCR’s 98.56% detection rate and proving that the LAMP assay is more sensitive and easier to implement in routine settings. The LAMP assay requires only 64 μM calcein, 1 mM MnCl₂, and 5 mM MgSO₄, making it accessible and cost-effective for small-scale laboratories focused on food safety.
The optimized LAMP assay's simplicity and effectiveness could make it an essential tool for preventing foodborne outbreaks in the Philippines. Pavon and Balaga aim to develop this protocol into an accessible test kit and improve its use to detect other Salmonella strains in other food samples, ultimately enhancing public health and agricultural safety standards.
SOURCES CITED
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