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

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Title:Point-of-Care Electroflotation of Dispersed, Low Tolerance Pathogens Improves Detection Rates by Loop Mediated Isothermal Amplification.
Authors:Diaz, Lena M.
Contributors:Molecular Biosciences & Bioeng (department)
Keywords:agricultural diagnostics
field testing
food pathogens
sample preparation
molecular diagnostics
show 1 morenucleic acid amplification
show less
Date Issued:Dec 2017
Publisher:University of Hawaiʻi at Mānoa
Abstract: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.
Description:M.S. Thesis. University of Hawaiʻi at Mānoa 2017.
URI:http://hdl.handle.net/10125/62559
Rights:All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections: M.S. - Molecular Biosciences and Bioengineering


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