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Chin Sci Bull. 2013 Apr;58(10):1162-1168.

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

Author information

1
Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA ; Diagenetics, Inc, Honolulu, HI 96822, USA.
2
Program for Appropriate Technologies in Health (PATH), Seattle, WA 98109, USA.
3
Human Nutrition, Food, and Animal Science, University of Hawaii at Manoa, Honolulu, HI 96822, USA.
4
Blood Cell Storage Inc (BCSI), Seattle, WA 98109, USA.
5
Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA.

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.

KEYWORDS:

DNA; LAMP; assimilating probe; biosensor; food safety; molecular diagnostics

PMID:
25477717
PMCID:
PMC4251779

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