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Biol Eng Trans. 2011;4(2):69-80.

Non-Instrumented Nucleic Acid Amplification (NINA) for Rapid Detection of Ralstonia solanacearum Race 3 Biovar 2.

Author information

1
Laboratory Manager, Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, Hawaii.
2
Technical Officer/Portfolio Leader, Center for POC for Global Health, Program for Appropriate Technologies in Health (PATH), Seattle, Washington.
3
Product Development Technician, Center for POC for Global Health, Program for Appropriate Technologies in Health (PATH), Seattle, Washington.
4
Product Development Officer, Center for POC for Global Health, Program for Appropriate Technologies in Health (PATH), Seattle, Washington.
5
Director, Center for POC for Global Health, Program for Appropriate Technologies in Health (PATH), Seattle, Washington.
6
Professor, Department of Plant and Environmental Protection Sciences.
7
ASABE Member, Associate Professor, Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, Hawaii.

Abstract

We report on the use of a non-instrumented device for the implementation of a loop-mediated amplification (LAMP) based assay for the select-agent bacterial-wilt pathogen Ralstonia solanacearum race 3 biovar 2. Heat energy is generated within the device by the exothermic hydration of calcium oxide, and the reaction temperature is regulated by storing latent energy at the melting temperature of a renewable lipid-based engineered phase-change material. Endpoint detection of the LAMP reaction is achieved without opening the reaction tube by observing the fluorescence of an innovative FRET-based hybridization probe with a simple custom fluorometer. Non-instrumented devices could maintain reactions near the design temperature of 63°C for at least an hour. Using this approach DNA extracted from the pathogen could be detected at fewer than ten copies within a 25 μL reaction mix, illustrating the potential of these technologies for simple, powerful agricultural diagnostics in the field. Furthermore, the assay was just as reliable when implemented in a tropical environment at 31°C as it was when implemented in an air-conditioned lab maintained at 22°C, illustrating the potential value of the technology for field conditions in the tropics and subtropics.

KEYWORDS:

Agricultural diagnostics; Assimilating probe; Bacterial wilt; Biosensor; DNA; LAMP

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