Point-of-Care Electroflotation of Dispersed, Low Tolerance Pathogens Improves Detection Rates by Loop Mediated Isothermal Amplification.

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2017-12
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Diaz, Lena M.
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Molecular Biosciences & Bioeng
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Molecular diagnostic systems are becoming increasingly portable enabling rapid direct detection of pathogens in the field. However, trace contaminations of pathogens on food and in the environment remain notoriously difficult to detect, eluding the most sensitive molecular methods. For example gene-based assays typically test sample volumes of 1-5 μL, so that a single replicate of even a robust assay is statistically unlikely to detect pathogens at levels below 102-103 CFU/mL. To address the logistical requirements for successfully detecting pathogens dispersed on an ecological scale, we have developed a portable point-of-care (POC) sample preparation system using electroflotation (EF) to preferentially recover pathogenic organisms dispersed in hundreds of milliliters by concentrating samples into small (μL) assay formats. Electrolysis reactions, supported on custom designed platinum coated titanium electrodes, generate hydrogen and oxygen micro bubbles that impel and displace suspended cells into a recovered concentrate. Electrolysis conditions and durations were controlled with a custom AndroidOS application, with a system designed to concentrate cells suspended in 380 mL of phosphate buffer (0.1 M) sample into a small user defined concentrate volume in 30 minutes or less. To enhance viable cell recovery, variable concentrations of Pluronic-F68 (0.01, 0.1 g L-1) and chitosan oligosaccharide (0.01, 0.1 g L-1) were added to the sample media containing 102, 103 or 104 CFU/mL of Escherichia coli (E. coli) 25922 to produce shear protected flocs. EF processes were varied to include 10, 15 and 20 minutes of sample concentration at high and low turbulence flotation conditions. Evaluation of detection limits was conducted with and without EF treatment in a standardized loop mediated isothermal amplification (LAMP) assay targeting a single-copy gene (glycerate kinase) in E. coli 25922. By this method reliable detection (~>95 %) of E. coli 25922 by LAMP was achieved at concentrations down to 102 CFU / mL, representing an improvement in the detection limit of 3 orders of magnitude relative to untreated control samples.
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agricultural diagnostics, field testing, food pathogens, sample preparation, molecular diagnostics, nucleic acid amplification
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