Abstract
We report on the use of a novel non-instrumented platform to enable a Loop Mediated isothermal Amplification (LAMP) based assay for Salmonella enterica. Heat energy is provided by addition of a small amount (<150 g) of boiling water, and the reaction temperature is regulated by storing latent energy at the melting temperature of a lipid-based engineered phase change material. Endpoint classification of the reaction is achieved without opening the reaction tube by observing the fluorescence of sequence-specific FRET-based assimilating probes with a simple handheld fluorometer. At or above 22°C ambient temperature the non-instrumented devices could maintain reactions above a threshold temperature of 61°C for over 90 min-significantly longer than the 60 min reaction time. Using the simple format, detection limits were less than 20 genome copies for reactions run at ambient temperatures ranging from 8 to 36°C. When used with a pre-enrichment step and non-instrumented DNA extraction device, trace contaminations of Salmonella in milk close to 1 CFU/mL could be reliably detected. These findings illustrate that the non-instrumented amplification approach is a simple, viable, low-cost alternative for field-based food and agricultural diagnostics or clinical applications in developing countries.
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Kubota, R., Labarre, P., Weigl, B.H. et al. Molecular diagnostics in a teacup: Non-Instrumented Nucleic Acid Amplification (NINA) for rapid, low cost detection of Salmonella enterica . Chin. Sci. Bull. 58, 1162–1168 (2013). https://doi.org/10.1007/s11434-012-5634-9
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DOI: https://doi.org/10.1007/s11434-012-5634-9