A rapid on-site loop-mediated isothermal amplification technology as an early warning system for the detection of Shiga toxin-producing Escherichia coli in water

Author:

Alfahl Zina12ORCID,Biggins Sean31,Higgins Owen31,Chueiri Alexandra31,Smith Terry J.31,Morris Dearbháile12,O'Dwyer Jean45,Hynds Paul D.64,Burke Liam P.12,O’Connor Louise13

Affiliation:

1. Centre for One Health, Ryan Institute, University of Galway, Galway, Ireland

2. Antimicrobial Resistance and Microbial Ecology Group, School of Medicine, University of Galway, Galway, Ireland

3. Molecular Diagnostics Research Group, College of Science and Engineering, University of Galway, Galway, Ireland

4. Irish Centre for Research in Applied Geosciences (iCRAG), University College Dublin, Dublin, Ireland

5. School of Biological, Earth and Environmental Sciences, University College Cork, Cork, Ireland

6. Environmental Sustainability and Health Institute, Technological University Dublin, Dublin, Ireland

Abstract

Shiga toxin-producing Escherichia coli (STEC) is an important waterborne pathogen capable of causing serious gastrointestinal infections with potentially fatal complications, including haemolytic–uremic syndrome. All STEC serogroups harbour genes that encode at least one Shiga toxin (stx1 and/or stx2), which constitute the primary virulence factors of STEC. Loop-mediated isothermal amplification (LAMP) enables rapid real-time pathogen detection with a high degree of specificity and sensitivity. The aim of this study was to develop and validate an on-site portable diagnostics workstation employing LAMP technology to permit rapid real-time STEC detection in environmental water samples. Water samples (n=28) were collected from groundwater wells (n=13), rivers (n=12), a turlough (n=2) and an agricultural drain (n=1) from the Corrib catchment in Galway. Water samples (100 ml) were passed through a 0.22 µm filter, and buffer was added to elute captured cells. Following filtration, eluates were tested directly using LAMP assays targeting stx1, stx2 and E. coli phoA genes. The portable diagnostics workstation was used in field studies to demonstrate the on-site testing capabilities of the instrument. Real-time PCR assays targeting stx1 and stx2 genes were used to confirm the results. The limit of detection for stx1, stx2 and phoA LAMP assays were 2, 2 and 6 copies, respectively. Overall, stx1, stx2 and phoA genes were detected by LAMP in 15/28 (53.6 %), 9/28 (32.2 %) and 24/28 (85.7 %) samples, respectively. For confirmation, the LAMP results for stx1 and stx2 correlated perfectly (100 %) with those obtained using PCR. The portable diagnostics workstation exhibited high sensitivity throughout the on-site operation, and the average time from sample collection to final result was 40 min. We describe a simple, transferable and efficient diagnostic technology for on-site molecular analysis of various water sources. This method allows on-site testing of drinking water, enabling evidence-based decision-making by public health and water management authorities.

Funder

Environmental Protection Agency

Publisher

Microbiology Society

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