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Simultaneous detection of various pathogenic Escherichia coli in water by sequencing multiplex PCR amplicons

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Abstract

Waterborne diseases due to pathogen contamination in water are a serious problem all over the world. Accurate and simultaneous detection of pathogens in water is important to protect public health. In this study, we developed a method to simultaneously detect various pathogenic Escherichia coli by sequencing the amplicons of multiplex PCR. Our newly designed multiplex PCR amplified five genes for pathogenic E. coli (uidA, stx1, stx2, STh gene, and LT gene). Additional two PCR assays (for aggR and eae) were also designed and included in the amplicon sequencing analysis. The same assays were also used for digital PCR (dPCR). Strong positive correlations were observed between the sequence read count and the dPCR results for most of the genes targeted, suggesting that our multiplex PCR-amplicon sequencing approach could provide quantitative information. The method was also successfully applied to monitor the level of pathogenic E. coli in river water and wastewater samples. The approach shown here could be expanded by targeting genes for other pathogens.

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All data generated or analyzed during this study are included in this published article [and its supplementary information files].

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Acknowledgements

We thank Professor Tetsuya Hayashi and Professor Yoshitoshi Ogura for their advice on experimental design. We also thank Tomoko Fukudome, Yasuhiko Kawahara at the Miyazaki Prefectural Institute of Public Health and Environment for providing us with a DNA standard solution containing the target gene. The Bioengineering Lab. provided various information and suggestions for the sequencing data analysis.

Funding

This publication was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP 21H0362021.

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Authors and Affiliations

  1. Department of Civil and Environmental Engineering, Faculty of Engineering, University of Miyazaki, Miyazaki, Japan

    Yoshihiro Suzuki, Hiroki Shimizu, Shouichiro Tamai & Kei Nukazawa

  2. Department of Biological Functions Engineering, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu, 808-0196, Japan

    Yuki Hoshiko & Toshinari Maeda

  3. Present address: Division of Microbiology, Department of Infectious Medicine, Kurume University School of Medicine, 67 Asahi-Machi, Kurume-City, Fukuoka, 830-0011, Japan

    Yuki Hoshiko

  4. Department of Animal and Grassland Sciences, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan

    Atsushi Iguchi

  5. Center for Climate Change Adaptation, National Institute for Environmental Studies, Ibaraki, Japan

    Yoshifumi Masago

  6. Department of Soil, Water, and Climate, University of Minnesota, Falcon Heights, MN, USA

    Satoshi Ishii

  7. Bio Technology Institute, University of Minnesota, Falcon Heights, MN, USA

    Satoshi Ishii

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  1. Yoshihiro Suzuki
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Contributions

Conceptualization, supervision, investigation, writing—original draft, Y.S.; investigation, formal analysis, methodology, validation, H.S.; visualization, writing—review and editing, S.T.; methodology, Y.H., T.M.; validation, writing—review and editing, K.N; validation, A.I, Y.M.; formal analysis, software, validation, visualization, writing—review and editing, S.I. funding acquisition, Y.S. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Yoshihiro Suzuki.

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Suzuki, Y., Shimizu, H., Tamai, S. et al. Simultaneous detection of various pathogenic Escherichia coli in water by sequencing multiplex PCR amplicons. Environ Monit Assess 195, 264 (2023). https://doi.org/10.1007/s10661-022-10863-6

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