Rapid on-site monitoring of Legionella pneumophila in cooling tower water using a portable microfluidic system

Yamaguchi, Naohito; Tokunaga, Yusuke; Goto, Satoko; Fujii, Yudai; Banno, Fumiya; Edagawa, Akiko

doi:10.1038/s41598-017-03293-9

Cited by 31 publications

(19 citation statements)

References 35 publications

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“…LP3IIG2 shows compatible results to be applied in the specific L. pneumophila detection in water samples. This antibody can be applied in different methods to detect L. pneumophila (Yamaguchi et al, 2017;Yanez et al, 2005), improving specificity of the method. Continuing development by sourcing further monoclonal antibodies to increase the range of detection, automatization, and further optimization will improve the detection.…”

Section: Discussionmentioning

confidence: 99%

Antibody test for Legionella pneumophila detection

Párraga-Niño

Quero

Uría

et al. 2018

Diagnostic Microbiology and Infectious Disease

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Legionella pneumophila is responsible for Legionnaires' disease (LD). Its detection in both environmental and clinical samples is mainly performed by culture plate method which requires up to 10days to obtain results. Nowadays, there are commercial antibodies against this bacterium, but they have not been tested against all subgroups of L. pneumophila sg 1 or serogroups 1-16 or their cross-reactions with other non-Legionella bacteria. Indeed, many of these antibodies became available when only 8 serogroups of L. pneumophila had been described. We tested 7 antibodies and found that 2 (Mab 8/5 and OBT) specifically detected all the subgroups of L. pneumophila sg 1, one without cross-reactions (Mab8/5). Moreover, the LP3IIG2 antibody detected almost all serogroups tested with lower rates of cross-reactivity, resulting in a specific sensitive antibody for the detection of L. pneumophila. LP3IIG2 presented higher rate of cross-reactivity against respiratory non-Legionella isolates, thereby contraindicating its clinical applicability.

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Section: Discussionmentioning

confidence: 99%

Antibody test for Legionella pneumophila detection

Párraga-Niño

Quero

Uría

et al. 2018

Diagnostic Microbiology and Infectious Disease

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“…2) were constructed using rapid prototyping and replica-molding techniques. 21) The depth of the microchannel was 15 µm and the width was 100 or 500 µm.…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we applied our microfluidic device 21) to determination of the number of bacterial cells in freshwater without intensive labor; this device was originally designed for on-chip fluorescent staining and semi-automated counting of target microbial cells with fluorescent antibody-staining. In conjunction, we used a self-made portable system for rapid on-site monitoring of bacterial cells 22) to enumerate the total or physiologically active microbial cells flowing in the microchannel.…”

Section: Introductionmentioning

confidence: 99%

Rapid On-Site Monitoring of Bacteria in Freshwater Environments Using a Portable Microfluidic Counting System

Yamaguchi

Fujii

2020

Biological & Pharmaceutical Bulletin

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Freshwater environments and natural water parks are important as recreation areas; however, people enjoying recreation at the river-or lake-side are sometimes infected with pathogenic microbes. Microbiological monitoring is fundamental for the routine evaluation of water quality. Fluorescent staining techniques are regarded as among the most useful rapid microbiological methods; however, preparation of samples for fluorescence microscopy is often labor-intensive, and one usually has to take the samples to a laboratory for measurement, which often alters the culturability of bacteria in the samples. These factors have created demand for a rapid and simple method of bacterial quantification in freshwater that can be performed on-site. In this study, we applied our microfluidic device, which was originally designed for on-chip fluorescent staining and semi-automated counting of target microbial cells with fluorescent antibody-staining, to enumerate bacterial cells in freshwater. This was combined with a self-made portable system for rapid on-site monitoring of the bacterial cells. Numbers of both esterase-active bacteria and total bacteria in pond water samples could be successfully determined by on-chip staining with 6-carboxyfluorescein diacetate and SYBR Green II, respectively, using the portable microfluidic counting system. The counting was completed within 1 h (30 min for pre-filtration of freshwater and 30 min for on-chip staining and counting). These results indicate that rapid and accurate counting of bacterial cells in freshwater can be performed and this technique could be applied for "on-site first screening" purposes in microbial quality control of freshwater.

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“…These would have the flexibility for use in combination with various observation or detection techniques: for example, counting and characterization of stained microbes using epifluorescence microscopy (Nakajima et al ; Hara and Zhang ; Murata and Zhang ), and flow cytometry (Chen and Li ; Besmer et al ; Buysschaert et al ). Combinations of fluorescent staining with other methods also have been developed (Bisson et al ; Park et al ; Choi et al ; Yamaguchi et al ; Sorensen et al ; Swanson and Huffman ). Although the use of fluorescent staining has been reported as above, the staining conditions appear to be determined by trial and error.…”

Section: Introductionmentioning

confidence: 99%

Optimal fluorescent‐dye staining time for the real‐time detection of microbes: a study of Saccharomyces cerevisiae

et al. 2020

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Aims To provide information on the time‐dependent behaviour of microbe staining by fluorescent dyes in the order of seconds, which is important in terms of the recent rapid and online techniques for microbe measurements and/or environmental microbe analysis. Methods and Results For combinations of yeast (Saccharomyces cerevisiae) and typical dyes, including DAPI (4′,6‐diamidino‐2‐phenylindole) and Auramine‐O, a suspension of yeast cells in ultrapure water was injected into a dye solution in a micro cuvette placed inside a spectrofluorometer and the fluorescence intensity of the resulting solution was measured at 1 s intervals, starting immediately after the mixing and continued until the time for the maximum intensity using various concentrations of yeast and dyes. The relaxation time τ, which corresponds to ~63·2% of the maximum fluorescence intensity, was shown to decrease to below 1 s with increasing DAPI concentration, whereas it remained constant for 2–3 s with increasing Auramine‐O concentration, for example at a yeast concentration of 100 µg ml−1. Conclusions For the conditions of yeast >10 µg ml−1, DAPI >1 µg ml−1 and Auramine‐O >0·1 µg ml−1, τ could be adjusted to below 5 s to achieve a rapid and stable staining. Significance and Impact of the Study Design and operating conditions for rapid and online measurements of microbes can be optimized.

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Rapid on-site monitoring of Legionella pneumophila in cooling tower water using a portable microfluidic system

Cited by 31 publications

References 35 publications

Antibody test for Legionella pneumophila detection

Antibody test for Legionella pneumophila detection

Rapid On-Site Monitoring of Bacteria in Freshwater Environments Using a Portable Microfluidic Counting System

Optimal fluorescent‐dye staining time for the real‐time detection of microbes: a study of Saccharomyces cerevisiae

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