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

Abstract

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.

Whole-saliva Proteolysis and Its Impact on Salivary Diagnostics

“…It has been recognized that saliva is a readily available biofluid that meets the demands for non-invasive, stress-free, and inexpensive collection, offering distinctive advantages over blood collection (Mandel and Wotman, 1976). Recent evidence for saliva's diagnostic capabilities include the identification of MMP-8 and -9 and osteoprotegrin as biomarkers for periodontal disease (Ramseier et al, 2009), changes in salivary inflammatory cytokine profiles in asthma exacerbations (Blicharz et al, 2009), salivary detection of HIV-1 (Liu et al, 2011), the correlation between salivary transcriptome markers KRAS, BMD3L2, ACRV1, and DPM1 and pancreatic cancer (Zhang et al, 2010), and salivary C-reactive protein, myoglobin, and myeloperoxidase biomarkers to detect acute myocardial infarction (Floriano et al, 2009). The attractiveness of using WS for diagnostic purposes, however, has frequently superseded the need to preserve the stability of this body fluid during and/or after collection.…”

Microfluidic “Pouch” Chips for Immunoassays and Nucleic Acid Amplification Tests

“…Pouches P 2 and P 3 are depressed in succession to wash the membrane-bound nucleic acid. For more detailed description regarding the structures and operations related to NA isolation, PCR, and detection, see Wang et al [19], Chen et al [5], and related work of Kim et al [22,23], Liu et al [24,25], and Mauk et al [26].…”

A disposable, integrated loop-mediated isothermal amplification cassette with thermally actuated valves

“…As an example of a RNA target, we used the RNA control included in the reverse transcription, loop-mediated, isothermal amplification (RT-LAMP) kit provided by the Eiken Chemical Co. In a separate work, we used the LAMP process to amplify and detect HIV RNA (Liu et al 2011 The LAMP cassette: a exploded view of the cassette. The cassette consists of three solvent-bonded layers of polycarbonate.…”