An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases

A simple, point of care, inexpensive, disposable cassette for the detection of nucleic acids extracted from pathogens was designed, constructed, and tested. The cassette utilizes a single reaction chamber for isothermal amplification of nucleic acids. The chamber is equipped with an integrated, flow...

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Bibliographic Details
Published in:Analyst (London) 2011-05, Vol.136 (10), p.2069-2076
Main Authors: CHANGCHUN LIU, GEVA, Eran, MAUK, Michael, XIANBO QIU, ABRAMS, William R, MALAMUD, Daniel, CURTIS, Kelly, OWEN, S. Michele, BAN, Haim H
Format: Article
Language:English
Subjects:
Amplification
Analytical chemistry
Cassettes
Chambers
Chemistry
Exact sciences and technology
HIV
HIV Infections - diagnosis
HIV-1 - genetics
Humans
Inhibitors
Membranes
Microfluidic Analytical Techniques - methods
Nucleic Acid Amplification Techniques - instrumentation
Nucleic Acid Amplification Techniques - methods
Nucleic acids
Point-of-Care Systems
Ribonucleic acids
RNA, Viral - analysis
RNA, Viral - isolation & purification
Saliva - virology
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Summary:A simple, point of care, inexpensive, disposable cassette for the detection of nucleic acids extracted from pathogens was designed, constructed, and tested. The cassette utilizes a single reaction chamber for isothermal amplification of nucleic acids. The chamber is equipped with an integrated, flow-through, Flinders Technology Associates (Whatman FTA®) membrane for the isolation, concentration, and purification of DNA and/or RNA. The nucleic acids captured by the membrane are used directly as templates for amplification without elution, thus simplifying the cassette's flow control. The FTA membrane also serves another critical role-enabling the removal of inhibitors that dramatically reduce detection sensitivity. Thermal control is provided with a thin film heater external to the cassette. The amplification process was monitored in real time with a portable, compact fluorescent reader. The utility of the integrated, single-chamber cassette was demonstrated by detecting the presence of HIV-1 in oral fluids. The HIV RNA was reverse transcribed and subjected to loop-mediated, isothermal amplification (LAMP). A detection limit of less than 10 HIV particles was demonstrated. The cassette is particularly suitable for resource poor regions, where funds and trained personnel are in short supply. The cassette can be readily modified to detect nucleic acids associated with other pathogens borne in saliva, urine, and other body fluids as well as in water and food.
ISSN:0003-2654
1364-5528
DOI:10.1039/c1an00007a