Electroflotation of Escherichia coli Improves Detection Rates by Loop-mediated Isothermal Amplification

摘要

The power of portable molecular diagnostic systems for the detection of pathogenic microorganisms in food and environmental samples is largely limited by small assay volumes (typically 1-5 uL), making direct detection of trace contamination (i.e. <104CFU / mL) unreliable. To improve detection limits for pathogens dispersed on an ecological scale, we have developed a portable point-of-care (POC) sample preparation system using electroflotation (EF) to recover small quantities of these organisms fromsamples of hundreds of milliliters. Electrolysis reactions, supported on platinum-coated titanium electrodes, generate hydrogen and oxygen micro-bubbles that impel and displace suspended cells into a recovered concentrate. Samples were prepared by inoculating 380 mL of sterilized phosphate buffer (0.1 M, pH 6.6) with stock culture of Escherichia coli (E. coli) ATCC 25922 to final concentrations ranging from 102 to 104CFU/mL. Samples were subjected to 10, 15, and 20 minute durations of EF treatment underhigh and low turbulent mixing conditions. We used a loop mediated amplification (LAMP) assay with primers targeting a single-copy gene (glycerate kinase) in generic E. coli to evaluate the effects of EF treatment on concentration and recovery of detectable cell material. LAMP failed to detect E. coli in all untreated (control) samples at concentrations below 104 CFU/mL, but was able to detect E. coli in 102 CFU/mL samples subjected to various conditions of EF treatment. Two-way ANOVA showed significantdifferences in detection rates between EF treatment durations for both high (p=0.0019) and low turbulence (p=0.002). Dunnett's multiple comparison tests identified 5 process conditions resulting insignificant (p<0.05) differences in detection between treatments and the control.