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

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.

Naked-Eye Detection of LAMP-Produced Nucleic Acids in Saliva using Chitosan-capped AuNPs in a Single-Tube Assay

“…45 Naked-eye detection of LAMP amplicons in purified samples using Chit-AuNPs A previous study has shown that directly incorporating water-soluble Chit in a LAMP reaction inhibits DNA amplification because positively charged Chit conjugates with negatively charged DNA or interferes with LAMP primer annealing. 36 This was further confirmed when we added either dissolved neat Chit, or Chit-AuNPs in their originally synthesized acidic environment (replaced 5μL of nuclease-free water with Chit or Chit-AuNPs) and included as well 0.5 μL of LAMP fluorescent dye (readable in the SYBR ® /FAM channel) for detection; in both cases, no amplification signal was observed (Fig. S1), as the pH of the final mixture (measured with a pH strip) is ~4.5 owing to the acetic acid addition.…”

“…Although Chit appears promising for DNA-binding combined with aggregation/stabilization and colorimetric detection, its compatibility with LAMP has been low. 36 To the best of our knowledge, Chit-capped AuNPs visual observation of LAMP products has not been reported yet. Overall, despite recent advances, the development of simple and economic colorimetric assays for naked-eye detection with high sensitivity still remains a challenge, i.e., detecting a few copies of the target in a crude sample with a low contamination risk (single-tube assay).…”

Naked-Eye Detection of LAMP-Produced Nucleic Acids in Saliva Using Chitosan-Capped AuNPs in a Single-Tube Assay

“…Moreover, it has been demonstrated to act both as a reducing and stabilizing agent in a simple AuNPs synthesis procedure based on chemical reduction in aqueous environments; this method does not require toxic solvents or extra reducing agents. Although Chit appears promising for DNA-binding combined with aggregation/stabilization and colorimetric detection, its compatibility with LAMP has been low . To the best of our knowledge, Chit-capped AuNPs visual observation of LAMP products has not yet been reported.…”

“…Although Chit appears promising for DNA-binding combined with aggregation/ stabilization and colorimetric detection, its compatibility with LAMP has been low. 37 To the best of our knowledge, Chitcapped AuNPs visual observation of LAMP products has not yet been reported. Overall, despite recent advances, the development of simple and economic colorimetric assays for naked-eye detection with high sensitivity remains a challenge.…”

A Microfluidic Chip for LAMP-based Multiplex Detection of Pathogen

“…The total genomic DNA was extracted by using kit, following the instruction from manufacturer. The targets gene used for these bacterial detections were ecolc 3109_1[18], nuc [19]and opr [20], respectively. The primers were designed as previously reported and listed in Table1.…”