Skip to content
1887

Microbiology Society logo Microbiology

Volume 170, Issue 8

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

Abstract

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.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): E. coli , LAMP , STEC , testing , toxins and water contamination
Funding
This study was supported by the:
  • Environmental Protection Agency (Award 2021-HE-1033)
    • Principal Award Recipient: ZinaAlfahl
© 2024 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001485
2024-08-07
2025-11-10

Metrics

Loading full text...

Full text loading...

/deliver/fulltext/micro/170/8/mic001485.html?itemId=/content/journal/micro/10.1099/mic.0.001485&mimeType=html&fmt=ahah

References

  1. Martinson JNV, Walk ST. Escherichia coli residency in the gut of healthy human adults. EcoSal Plus 2020; 9:9 [View Article] [PubMed]
     [Google Scholar]
  2. Pokharel P, Dhakal S, Dozois CM. The diversity of Escherichia coli pathotypes and vaccination strategies against this versatile bacterial pathogen. Microorganisms 2023; 11:344 [View Article]
     [Google Scholar]
  3. Kaper JB, Nataro JP, Mobley HLT. Pathogenic Escherichia coli. Nat Rev Microbiol 2004; 2:123–140 [View Article] [PubMed]
     [Google Scholar]
  4. Tarr PI, Gordon CA, Chandler WL. Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet 2005; 365:1073–1086 [View Article]
     [Google Scholar]
  5. Koutsoumanis K, Allende A, Alvarez‐Ordóñez A, Bover‐Cid S, Chemaly M et al. Pathogenicity assessment of Shiga toxin‐producing Escherichia coli (STEC) and the public health risk posed by contamination of food with STEC. EFS2 2020; 18:e05967 [View Article]
     [Google Scholar]
  6. Ray R, Singh P. Prevalence and implications of Shiga toxin-producing E. coli in farm and wild ruminants. Pathogens 2022; 11:1332 [View Article] [PubMed]
     [Google Scholar]
  7. Holme R. Drinking water contamination in Walkerton, Ontario: positive resolutions from a tragic event. Water Sci Technol 2003; 47:1–6 [PubMed]
     [Google Scholar]
  8. Cobbold RN, Hancock DD, Rice DH, Berg J, Stilborn R et al. Rectoanal junction colonization of feedlot cattle by Escherichia coli O157:H7 and its association with supershedders and excretion dynamics. Appl Environ Microbiol 2007; 73:1563–1568 [View Article] [PubMed]
     [Google Scholar]
  9. McAllister TA, Topp E. Role of livestock in microbiological contamination of water: commonly the blame, but not always the source. Anim Front 2012; 2:17–27 [View Article]
     [Google Scholar]
  10. Topalcengiz Z, Jeamsripong S, Spanninger PM, Persad AK, Wang F et al. Survival of Shiga toxin-producing Escherichia coli in various wild animal feces that may contaminate produce. J Food Prot 2020; 83:1420–1429 [View Article] [PubMed]
     [Google Scholar]
  11. HSE - Health Protection Surveillance Centre Gastroenteric and Zoonotic Diseases in Ireland, 2022. Dublin: HPSC; 2024
  12. Andrade L, Boudou M, Hynds P, Chique C, Weatherill J et al. Spatiotemporal dynamics of Escherichia coli presence and magnitude across a national groundwater monitoring network, Republic of Ireland, 2011-2020. Sci Total Environ 2022; 840:156311 [View Article] [PubMed]
     [Google Scholar]
  13. Chique C, Hynds P, Burke LP, Morris D, Ryan MP et al. Contamination of domestic groundwater systems by verotoxigenic Escherichia coli (VTEC), 2003-2019: a global scoping review. Water Res 2021; 188:116496 [View Article] [PubMed]
     [Google Scholar]
  14. Chique C, Hynds PD, Andrade L, Burke L, Morris D et al. Cryptosporidium spp. in groundwater supplies intended for human consumption - a descriptive review of global prevalence, risk factors and knowledge gaps. Water Res 2020; 176:115726 [View Article] [PubMed]
     [Google Scholar]
  15. Brehony C, Cullinan J, Cormican M, Morris D. Shiga toxigenic Escherichia coli incidence is related to small area variation in cattle density in a region in Ireland. Sci Total Environ 2018; 637–638:865–870 [View Article] [PubMed]
     [Google Scholar]
  16. EPA Focus on Private Water Supplies; 2017
  17. Sattar AA, Good CR, Saletes M, Brandão J, Jackson SK. Endotoxin as a marker for water quality. Int J Environ Res Public Health 2022; 19:19 [View Article] [PubMed]
     [Google Scholar]
  18. Gould LH, Mody RK, Ong KL, Clogher P, Cronquist AB et al. Increased recognition of non-O157 Shiga toxin-producing Escherichia coli infections in the United States during 2000-2010: epidemiologic features and comparison with E. coli O157 infections. Foodborne Pathog Dis 2013; 10:453–460 [View Article] [PubMed]
     [Google Scholar]
  19. Parsons BD, Zelyas N, Berenger BM, Chui L. Detection, characterization, and typing of Shiga toxin-producing Escherichia coli. Front Microbiol 2016; 7:478 [View Article] [PubMed]
     [Google Scholar]
  20. Dong HJ, Cho AR, Hahn TW, Cho S. Development of a multiplex loop-mediated isothermal amplification assay to detect Shiga toxin-producing Escherichia coli in cattle. J Vet Sci 2014; 15:317–325 [View Article] [PubMed]
     [Google Scholar]
  21. Alfahl Z, O’ Connor L, Morris D, Smith TJ, Burgess C et al. A novel enrichment-free, low-volume filtration and rapid lysis method (Elr) in combination with real-time PCR for detection of Shiga toxin-producing Escherichia coli (STEC) in water. SSRN 2024 [View Article]
     [Google Scholar]
  22. Stratakos AC, Linton M, Millington S, Grant IR. A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenic Escherichia coli in beef and bovine faeces. J Appl Microbiol 2017; 122:817–828 [View Article] [PubMed]
     [Google Scholar]
  23. Perelle S, Dilasser F, Grout J, Fach P. Detection by 5’-nuclease PCR of Shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world’s most frequent clinical cases. Mol Cell Probes 2004; 18:185–192 [View Article] [PubMed]
     [Google Scholar]
  24. Cleary E, Boudou M, Garvey P, Aiseadha CO, McKeown P et al. Spatiotemporal dynamics of sporadic Shiga toxin-producing Escherichia coli Enteritis, Ireland, 2013-2017. Emerg Infect Dis 2021; 27:2421–2433 [View Article] [PubMed]
     [Google Scholar]
  25. Hynes R, Alfahl Z, O’Connor L, De Bock F, Burgess C et al. Longitudinal monitoring of Shiga toxin-producing Escherichia coli (STEC) concentrations in surface waters and groundwater supplies within an Irish catchment. European Geosciences Union 2024; Abstract 16975; 2024
  26. Terajima J, Izumiya H, Hara-Kudo Y, Ohnishi M. Shiga toxin (Verotoxin)-producing Escherichia coli and foodborne disease: a review. Food Saf 2017; 5:35–53 [View Article] [PubMed]
     [Google Scholar]
  27. Gadkar VJ, Goldfarb DM, Gantt S, Tilley PAG. Real-time detection and monitoring of loop mediated amplification (LAMP) reaction using self-quenching and de-quenching fluorogenic probes. Sci Rep 2018; 8:5548 [View Article] [PubMed]
     [Google Scholar]
  28. Foo PC, Nurul Najian AB, Muhamad NA, Ahamad M, Mohamed M et al. Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample. BMC Biotechnol 2020; 20:34 [View Article] [PubMed]
     [Google Scholar]
  29. ECDC STEC infection annual epidemiological report for 2022. European Centre for Disease Prevention and Control; 2022
  30. Martínez-Castillo A, Muniesa M. Implications of free Shiga toxin-converting bacteriophages occurring outside bacteria for the evolution and the detection of Shiga toxin-producing Escherichia coli. Front Cell Infect Microbiol 2014; 4:46 [View Article] [PubMed]
     [Google Scholar]
  31. Higgins O, Chueiri A, O’Connor L, Lahiff S, Burke L et al. Portable differential detection of CTX-M ESBL gene variants, bla(CTX-M-1) and bla(CTX-M-15), from Escherichia coli isolates and animal fecal samples using loop-primer endonuclease cleavage loop-mediated isothermal amplification. Microbiol Spectr 2023; 11:e0331622
     [Google Scholar]
  32. Lee S, Khoo VSL, Medriano CAD, Lee T, Park S-Y et al. Rapid and in-situ detection of fecal indicator bacteria in water using simple DNA extraction and portable loop-mediated isothermal amplification (LAMP) PCR methods. Water Res 2019; 160:371–379 [View Article] [PubMed]
     [Google Scholar]
  33. Martzy R, Kolm C, Brunner K, Mach RL, Krska R et al. A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Enterococcus spp. in water. Water Res 2017; 122:62–69 [View Article] [PubMed]
     [Google Scholar]
  34. Wang F, Jiang L, Ge B. Loop-mediated isothermal amplification assays for detecting Shiga toxin-producing Escherichia coli in ground beef and human stools. J Clin Microbiol 2012; 50:91–97 [View Article] [PubMed]
     [Google Scholar]
  35. Wang F, Yang Q, Qu Y, Meng J, Ge B. Evaluation of a loop-mediated isothermal amplification suite for the rapid, reliable, and robust detection of Shiga toxin-producing Escherichia coli in produce. Appl Environ Microbiol 2014; 80:2516–2525 [View Article] [PubMed]
     [Google Scholar]
  36. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000; 28:E63 [View Article] [PubMed]
     [Google Scholar]
  37. Zhao X, Li Y, Wang L, You L, Xu Z et al. Development and application of a loop-mediated isothermal amplification method on rapid detection Escherichia coli O157 strains from food samples. Mol Biol Rep 2010; 37:2183–2188 [View Article] [PubMed]
     [Google Scholar]
  38. Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother 2009; 15:62–69 [View Article] [PubMed]
     [Google Scholar]
  39. Tanner NA, Evans TC. Loop-mediated isothermal amplification for detection of nucleic acids. Curr Protoc Mol Biol 2014; 105:14–15 [View Article] [PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.001485
Loading
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
Microbiology 170, 001485 (2024); https://doi.org/10.1099/mic.0.001485
/content/journal/micro/10.1099/mic.0.001485
/content/journal/micro/10.1099/mic.0.001485
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An error occurred
Approval was partially successful, following selected items could not be processed due to error