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) o...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.展开更多
基金Financial support at UH was provided by USDA-Hatch project HA-517NINA concepts and prototypes developed at PATH were supported by grants under the Health Innovations Portfolio,which is supported by the United States Agency for International Development,private foundations,and individual donors
文摘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.
