Molecular testing devices for on-site detection of <i>E. coli</i> in water samples

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Abstract

Escherichia coli ( E. coli) cells are present in fecal materials that can be the main source for disease‐causing agents in water. As a result, E. coli is recommended as a water quality indicator. We have developed an innovative platform to detect E. coli for monitoring water quality on-site by integrating paper-based sample preparation with nucleic acid isothermal amplification. The platform carries out bacterial lysis and DNA enrichment onto a paper pad through ball-based valves for fluid control, with no need of laboratory equipment, followed by loop-mediated isothermal amplification (LAMP) in a battery-operated coffee mug, and colorimetric detection. We have used the platform to detect E. coli in environmental water samples in about 1 h, with a limit of quantitation of 0.2 CFU/mL, and 3 copies per reaction. The platform was confirmed for detecting multiple E. coli strains, and for water samples of different salt concentrations. We validated the functions of the platform by analyzing recreational water samples collected near the Atlantic Ocean that contain different concentrations of salt and bacteria.

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Most cited references 48

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Loop-mediated isothermal amplification of DNA.

T. Notomi (2000)

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.

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Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.

Norihiro Tomita, Yasuyoshi Mori, Hidetoshi Kanda … (2008)

As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Critical to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification. This protocol details an improved simple visual detection system for the results of the LAMP reaction. In LAMP, a large amount of DNA is synthesized, yielding a large pyrophosphate ion by-product. Pyrophosphate ion combines with divalent metallic ion to form an insoluble salt. Adding manganous ion and calcein, a fluorescent metal indicator, to the reaction solution allows a visualization of substantial alteration of the fluorescence during the one-step amplification reaction, which takes 30-60 min. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.

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Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes.

Nathan A. Tanner, Yinhua Zhang, Thomas Evans (2015)

Nucleic acid amplification is the basis for many molecular diagnostic assays. In these cases, the amplification product must be detected and analyzed, typically requiring extended workflow time, sophisticated equipment, or both. Here we present a novel method of amplification detection that harnesses the pH change resulting from amplification reactions performed with minimal buffering capacity. In loop-mediated isothermal amplification (LAMP) reactions, we achieved rapid (<30 min) and sensitive (<10 copies) visual detection using pH-sensitive dyes. Additionally, the detection can be performed in real time, enabling high-throughput or quantitative applications. We also demonstrate this visual detection for another isothermal amplification method (strand-displacement amplification), PCR, and reverse transcription LAMP (RT-LAMP) detection of RNA. The colorimetric detection of amplification presented here represents a generally applicable approach for visual detection of nucleic acid amplification, enabling molecular diagnostic tests to be analyzed immediately without the need for specialized and expensive instrumentation.

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Author and article information

Contributors

Elise Morrison: elise.morrison@essie.ufl.edu

Todd Z. Osborne: osbornet@ufl.edu

Z. Hugh Fan: hfan@ufl.edu

Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2045-2322

Publication date (Electronic): 14 March 2023

Publication date PMC-release: 14 March 2023

Publication date Collection: 2023

Volume: 13

Electronic Location Identifier: 4245

Affiliations

[1 ]GRID grid.15276.37, ISNI 0000 0004 1936 8091, Interdisciplinary Microsystems Group, Department of Mechanical and Aerospace Engineering, , University of Florida, ; P.O. Box 116250, Gainesville, FL 32611 USA

[2 ]GRID grid.15276.37, ISNI 0000 0004 1936 8091, Department of Environmental Engineering Sciences, , University of Florida, ; P.O. Box 116580, Gainesville, FL 32611 USA

[3 ]GRID grid.15276.37, ISNI 0000 0004 1936 8091, Department of Molecular Genetics and Microbiology, , University of Florida, ; PO Box 100266, Gainesville, FL 32610 USA

[4 ]GRID grid.15276.37, ISNI 0000 0004 1936 8091, Whitney Laboratory of Marine Bioscience, , University of Florida, ; P.O. Box 116580, St. Augustine, FL 32080 USA

[5 ]GRID grid.15276.37, ISNI 0000 0004 1936 8091, Soil, Water, and Ecosystem Sciences Department, , University of Florida, ; P.O. Box 110290, Gainesville, FL 32611 USA

[6 ]GRID grid.15276.37, ISNI 0000 0004 1936 8091, J. Crayton Pruitt Family Department of Biomedical Engineering, , University of Florida, ; P.O. Box 116131, Gainesville, FL 32611 USA

Article

Publisher ID: 31208

DOI: 10.1038/s41598-023-31208-4

PMC ID: 10013241

PubMed ID: 36918634

SO-VID: 37a9767e-f991-42c0-8fa9-b19f00b7f54b

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 9 December 2022

Date accepted : 8 March 2023

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;

Award ID: R01AI155735

Award Recipient : Carlos Manzanas Z. Hugh Fan

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ScienceOpen disciplines: Uncategorized

Keywords: pathogens,water microbiology,diagnosis,biomedical engineering,mechanical engineering

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ScienceOpen disciplines: Uncategorized

Keywords: pathogens, water microbiology, diagnosis, biomedical engineering, mechanical engineering

Molecular testing devices for on-site detection of E. coli in water samples

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Hematology and Oncology Discovery

Most cited references 48

Loop-mediated isothermal amplification of DNA.

Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.

Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes.

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