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

• Published in: Microbiology (Society for General Microbiology) 2024-08, Vol.170 (8)
• Main Authors: Alfahl, Zina, Biggins, Sean, Higgins, Owen, Chueiri, Alexandra, Smith, Terry J, Morris, Dearbháile, O'Dwyer, Jean, Hynds, Paul D, Burke, Liam P, O'Connor, Louise
• Format: Article
• Language: English
• Subjects: Groundwater - microbiology; Method; Microbial Virulence and Pathogenesis; Molecular Diagnostic Techniques - instrumentation; Molecular Diagnostic Techniques - methods; Nucleic Acid Amplification Techniques - methods; Rivers - microbiology; Sensitivity and Specificity; Shiga Toxin 1 - genetics; Shiga-Toxigenic Escherichia coli - genetics; Shiga-Toxigenic Escherichia coli - isolation & purification; Water Microbiology
• ISSN: 1350-0872; 1465-2080; 1465-2080
• DOI: 10.1099/mic.0.001485

Shiga toxin-producing (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 ( and/or ), 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 ( =28) were collected from groundwater wells ( =13), rivers ( =12), a turlough ( =2) and an agricultural drain ( =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 , and 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 and genes were used to confirm the results. The limit of detection for , and LAMP assays were 2, 2 and 6 copies, respectively. Overall, , and 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 and 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.