title: Molecular diagnostics in a teacup: Non-Instrumented Nucleic Acid Amplification (NINA) for rapid, low cost detection ofSalmonella enterica.
abstract: We report on the use of a novel non-instrumented platform to enable a Loop Mediated isothermal Amplification (LAMP) based assay forSalmonella 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.
authors.affiliation: Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA ; Diagenetics, Inc, Honolulu, HI 96822, USA.
authors.forename: Ryo
authors.initials: R
authors.lastname: Kubota
authors.affiliation: Program for Appropriate Technologies in Health (PATH), Seattle, WA 98109, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Labarre
authors.affiliation: Program for Appropriate Technologies in Health (PATH), Seattle, WA 98109, USA.
authors.forename: Bernhard H
authors.initials: BH
authors.lastname: Weigl
authors.affiliation: Human Nutrition, Food, and Animal Science, University of Hawaii at Manoa, Honolulu, HI 96822, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Li
authors.affiliation: Blood Cell Storage Inc (BCSI), Seattle, WA 98109, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Haydock
authors.affiliation: Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Jenkins
keywords: DNA; LAMP; assimilating probe; biosensor; food safety; molecular diagnostics