Molecular diagnostics in a teacup:Non-Instrumented Nucleic Acid Amplification(NINA) for rapid,low cost detection of Salmonella enterica

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℃ ambient temperature the non-instrumented devices could maintain reactions above a threshold temperature of 61℃ 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℃.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.

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 se- quence-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.