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

Abstract

Abstract. The power of portable molecular diagnostic systems for detection of pathogenic microorganisms in food and environmental samples is largely limited by small assay volumes (typically 1 to 5 µL), making direct detection of trace contamination (i.e., <104 CFU mL-1) unreliable. To improve detection limits for pathogens dispersed on an ecological scale, we developed a portable point-of-care (POC) sample preparation system using electroflotation (EF) to recover small quantities of these organisms from samples of hundreds of milliliters. Electrolysis reactions, supported on platinum-coated titanium electrodes, generate hydrogen and oxygen microbubbles 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 ATCC 25922 to final concentrations ranging from 102 to 104 CFU mL-1. Samples were subjected to 10, 15, and 20 min durations of EF treatment under high and low turbulence conditions. We used a loop-mediated amplification (LAMP) assay with primers targeting a single-copy gene (glycerate kinase) in generic to evaluate the effects of EF treatment on concentration and recovery of detectable cell material. LAMP failed to detect in all untreated (control) samples at concentrations below 104 CFU mL-1 but was able to detect in 102 CFU mL-1 samples subjected to various conditions of EF treatment. Two-way ANOVA showed significant differences 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 five process conditions resulting in significant (p < 0.05) differences in detection between treatments and the control. Keywords: Biotechnology, Electrolysis, Food pathogens, Microbubbles, Molecular diagnostics, Pathogen detection, POC sample preparation.

Chemical stabilization of dispersed Escherichia coli for enhanced recovery with a handheld electroflotation system and detection by Loop-mediated Isothermal AMPlification

“…Subsequent a posteriori analysis ( Table 4 ) identified 0.01 L -1 and 0.1 g L -1 pluronic concentrations tested at 10 3 CFU/mL had significantly different detection rates of 85.18% and 92.59% respectively when compared to corresponding controls without pluronic (25.9%) ( Fig 4D ). This is a significant improvement from the ~25% detection rates that were observed in corresponding controls without pluronic and 0% detection rates without electroflotation [ 29 ].…”

“…The LAMP assay could detect dispersed E . coli present in quantities of 10 4 CFU/mL and 10 5 CFU/mL at rates of ~50% and 100% respectively [ 29 ]. Optimizing surfactant (Pluronic® F-68) and flocculant (chitosan) concentrations eventually allowed us to detect bacterial quantities of 10 2 CFU/mL at average rates of 96.3%-100%.…”

“…To target E . coli 25922 we used a previously characterized LAMP primer set [ 29 ] designated EcolC 3109_1 ( Table 1 ; [ 29 ]), targeting a conserved glycerate kinase coding region (EcolC 3109, Accession number: CP000946) of generic E . coli ATCC 8739.…”

“…Flotation of E . coli 25922 was carried out in a custom engineered handheld electroflotation (EF) cell with an approximate 400 mL capacity [ 29 ]. Electrolysis reactions were supported on inert platinum plated titanium electrode arrays.…”

“…In this report, we investigate the effects of adding chemical stabilizers (pluronic and chitosan) to enhance concentration, recovery and detection by LAMP of small quantities of dispersed bacterial contaminants by electroflotation (EF) performed in a hand-held automated system [ 29 ]. Additionally, a simple sample preparation procedure was developed so that recovered EF samples could be directly added to a LAMP assay without DNA purification or inhibition on LAMP reaction.…”