Abstract
Constraints related to sample preparation are some of the primary obstacles to widespread deployment of molecular diagnostics for rapid detection of trace quantities (≤103 CFU/mL) of food-borne pathogens. In this research, we report a sample preparation method using a novel handheld electroflotation system to concentrate and recover dilute quantities (102−103 CFU/mL) of Escherichia coli (E. coli) 25922 in artificially contaminated samples for reliable, rapid detection by loop-mediated isothermal amplification (LAMP). To protect suspended cells from shear stresses at bubble surfaces, a non-ionic surfactant (Pluronic-F68) and flocculant (chitosan oligosaccharide) were used to aggregate cells and reduce their surface hydrophobicity. Effective conditions for recovery were determined through multifactorial experiments including various concentrations of Pluronic-F68 (0.001, 0.01, 0.1, 1 g L-1), chitosan oligosaccharide (0.01, 0.1, 1, 10 g L-1), bacteria (102, 103, 104 CFU/mL E. coli 25922), recovery times (10, 15 and 20 minutes), and degrees of turbulent gas flux (“high” and “low”). The automated electroflotation system was capable of concentrating effectively all of the bacteria from a large sample (380 mL 0.1 M potassium phosphate buffer containing 102 CFU/mL E. coli) into a 1 mL recovered fraction in less than 30 minutes. This enabled detection of bacterial contaminants within 2 hours of collecting the sample, without a specialized laboratory facility or traditional enrichment methods, with at least a 2–3 order of magnitude improvement in detection limit compared to direct assay with LAMP.
Highlights
In the past 10 years food safety programs initiated by the United States Food and Drug Administration (FDA) including the Food Safety Modernization Act [1], Current Good Manufacturing Practices (CGMPs) [2] and Hazard Analysis Critical Control Point (HACCP) [3] imply that the generation of food safety guidance’s need to encompass the entire supply chain, from farm to table
We investigate the effects of adding chemical stabilizers to enhance concentration, recovery and detection by loop-mediated isothermal amplification (LAMP) of small quantities of dispersed bacterial contaminants by electroflotation (EF) performed in a hand-held automated system [29]
Using Dunnett’s multiple comparison a posteriori analysis, chitosan concentrations of 1g L-1 resulted in significant differences, but not complete inhibition, in mean threshold times compared to the control with no chitosan (Fig 2)
Summary
In the past 10 years food safety programs initiated by the United States Food and Drug Administration (FDA) including the Food Safety Modernization Act [1], Current Good Manufacturing Practices (CGMPs) [2] and Hazard Analysis Critical Control Point (HACCP) [3] imply that the generation of food safety guidance’s need to encompass the entire supply chain, from farm to table. Acknowledging that technological advances introduced a new imperative. Chemical stabilization of dispersed pathogens enhances recovery with a handheld electroflotation system decision to publish, or preparation of the manuscript
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