American Journal of Environmental Protection

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The Development and Application of DDPCR Technology on Quantification of Total Coliforms in Water

Received: 15 April 2020    Accepted: 30 April 2020    Published: 14 May 2020
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Abstract

In this research, the detection method for absolute quantification of total coliforms was established based on Droplet Digital Polymerase Chain Reaction (DDPCR) technology using lacZ as the target gene for coliform group detection. The experimental conditions (e.g. primer and probe concentrations, annealing temperatures, etc) were well optimized. Besides, the linear range, precision and limit of quantification (LOQ) of this method were investigated and evaluated. The results illustrated that the optimal primer concentration was 0.2 μmol/L, whereas the optimal probe concentration was 0.5 μmol/L. The optimal annealing temperature was 56°C. The linear relationship between the total coliform genome DNA concentrations derived from DDPCR and DNA fluorometer was quite good (R2 = 0.999). The linear range was 3.95 ~ 7.80 × 104 copies/20 μL DDPCR reaction system. The LOQ for total coliforms was single copy per reaction system. Practical applications using real water samples collected from water supply system in Macao illustrated that this innovative method possessed high efficiencies and capabilities. This is probably the first research using DDPCR technology to absolutely qualify and quantify total coliforms and successfully applied it in Macao water supply system. The achievements from this research could provide with significant values for setting-up the emergency mechanism of water pollution in early stage.

DOI 10.11648/j.ajep.20200902.11
Published in American Journal of Environmental Protection (Volume 9, Issue 2, April 2020)
Page(s) 22-30
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

Total Coliforms, DDPCR, Absolute Quantification, Water Supply, Water-borne Pathogen

References
[1] Ramírez-Castillo, F. Y., Loera-Muro, A., Jacques, M., Garneau, P., Avelar-González, F. J., Harel, J., and Guerrero-Barrera, A. L. (2015). Waterborne pathogens: detection methods and challenges. Pathogens, 4 (2): 307-334.
[2] McGinnis, S., Spencer, S., Firnstahl, A., Stokdyk, J., Borchardt, M., McCarthy, D. T., and Murphy, H. M. (2018). Human bacteroides and total coliforms as indicators of recent combined sewer overflows and rain events in urban creeks. Science of Total Environment, 630: 967-976.
[3] Mohammed, H., Hameed, I. A., and Seidu, R. (2018). Comparative predictive modelling of the occurrence of fecal indicator bacteria in a drinking water source in Norway. Science of Total Environment, 628-629: 1178-1190.
[4] Messner, M. J., Berger, P., and Javier, J. (2017). Total coliform and E. coli in public water systems using undisinfected ground water in the United Sates. International Journal of Hygiene and Environmental Health, 220 (4): 736-743.
[5] Wang, J., and Deng, Z. Q. (2019). Modeling and predicting fecal coliform bacteria levels in oyster harvest waters along Louisiana Gulf coast. Ecological Indicators, 101: 212-220.
[6] Noble, R. T., Moore, D. F., Leecaster, M. K., McGee, C. D., and Weisberg, S. B. (2003). Comparison of total coliform, fecal coliform and enterococcus bacterial indicator response for ocean recreational water quality testing. Water Research, 37 (7): 1637-1643.
[7] Maheux, A. F., Dion-Dupont, V., Bisson, M. A., Bouchard, S., and Rodriguez, M. J. (2014). Detection of Escherichia Coli colonies on confluent plates of chromogenic media used in membrane filtration. Journal of Microbiological Methods, 97: 51-55.
[8] Wang, D. L., and Fiessel, W. (2008). Evaluation of media for simultaneous enumeration of total coliform and Escherichia Coli in drinking water supplies by membrane filtration techniques. Journal of Environmental Sciences, 20 (3): 273-277.
[9] Eckner, K. F. (1998). Comparison of membrane filtration and multiple-tube fermentation by the Colilert and Enterolert methods for detection of waterborne coliform bacteria, Escherichia coli, and Enterococci used in drinking and bathing water quality monitoring in southern Sweden. Applied and Environmental Microbiology, 64 (8): 3079-3083.
[10] Wang, Z. D., Xiao, G. S., Zhou, N., Qi, W. H., Han, L., Ruan, Y., Guo, D. Q., and Zhou, H. (2015). Comparison of two methods for detection of fecal indicator bacteria used in water quality monitoring of the Three Gorges Reservoir. Journal of Environmental Sciences, 38: 42-51.
[11] Shelton, D. R., Higgins, J. A., Van Kessel, J. A. S., Pachepsky, Y. A., Belt, K., and Karns, J. S. (2004). Estimation of viable Escherichia Coli O157 in surface waters using enrichment in conjunction with immunological detection. Journal of Microbiological Methods, 58 (2): 223-231.
[12] Exum, N. G., Pisanic, N., Granger, D. A., Schwab, K. J., Detrick, B., Kosek, M., Egorov, A. I., Griffin, S. M., and Heaney, C. D. (2016). Use of pathogen-specific antibody biomarkers to estimate waterborne infections in population-based settings. Current Environmental Health Reports, 3: 322-334.
[13] Rompré, A., Servais. P., Baudart, J., de-Roubin, M. R., and Laurent, P. (2002). Detection and enumeration of coliforms in drinking water: current methods and emerging approaches. Journal of Microbiological Methods, 49 (1): 31-54.
[14] Van Poucke, S. O., and Nelis, H. J. (2000). Rapid detection of fluorescent and chemiluminescent total coliforms and Escherichia coli on membrane filters. Journal of Microbiological Methods, 42 (3): 233-244.
[15] Collins, S., Stevenson, D., Walker, J., and Bennett, A. (2017). Evaluation of Legionella real-time PCR against traditional culture for routine and public health testing of water samples. Journal of Applied Microbiology, 122: 1692-1703.
[16] Maheux, A. F., Huppé, V., Boissinot, M., Picard, F. J., Bissonnette, L., Bernier, J. L. T., and Bergeron, M. G. (2008). Analytical limits of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia Coli and total coliforms. Journal of Microbiological Methods, 75 (3): 506-514.
[17] Liu, Q., Zhang, X. L., Yao, Y. H., Jing, W. W., Liu, S. X., and Sui, G. D. (2018). A Novel microfluidic module for rapid detection of airborne and waterborne pathogens. Sensors and Actuators B: Chemical, 258: 1138-1145.
[18] Dao, T. N. T., Lee, E. Y., Koo, B., Jin, C. E., Lee, T. Y., and Shin, Y. (2018). A microfluidic enrichment platform with a recombinase polymerase amplification sensor for pathogen diagnosis. Analytical Biochemistry, 544: 87-92.
[19] Elsäßer, D., HO, J., Niessner, R., Tiehm, A., and Seidel, M. (2018). Heterogeneous asymmetric recombinase polymerase amplification (haRPA) for rapid hygiene control of large-volume water samples. Analytical Biochemistry, 546: 58-64.
[20] Martzy, R., Kolm, C., Brunner, K., Mach, R. L., Krska, R. Šinkovec, H., Sommer, R., Farnleitner, A. H., and Reischer, G. H. (2017). A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Enterococcus spp. in water. Water Research, 122: 62-69.
[21] Gunawardana, M., Chang, S., Jimenez, A., Holland-Moritz, D., Holland-Moritz, H., La VaL, T. P., Lund, C., Mullen, M., Olsen, J., Sztain, T. A., Yoo, J., Moss, J. A., and Baum, M. M. (2014). Isolation of PCR quality microbial community DNA from heavily contaminated environments. Journal of Microbiological Methods, 102: 1-7.
[22] Garofalo, C., Bancalari, E., Milanović, V., Cardinali, F., Osimani, A., Sardaro, M. L. S., Bottari, B., Bernini, V., Aquilanti, L., Clementi, F., Neviani, E., Gatti, M. (2017). Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR. International Journal of Food Microbiology, 242: 24-36.
[23] Lacout, A., Mone, Y., Franck, M., Marcy, P. Y., Mas, M., Veas, F., and Perronne, C. (2018). Blood cell disruption to significantly improve the borrelia PCR detection sensitivity in borreliosis in humans. Medical Hypothesis, 116: 1-3.
[24] Wang, Y. J., Zhu, S. Y., Hong, W. M., Wang, A. P., and Zuo, W. Y. (2017). A multiplex PCR for detection of six viruses in ducks. Journal of Virological Methods, 248: 172-176.
[25] Boehm, A. B., Van De Werfhorst, L. C., Griffith, J. F., Holden, P. A., Jay, J. A., Shanks, O. C., Wang, D., and Weisberg, S. B. (2013). Performance of forty-one microbial source tracking methods: a twenty-seven lab evaluation study. Water Research, 47 (18): 6812-6828.
[26] Griffith, J. F., and Weisberg, S. B. (2011). Challenges in implementing new technology for beach water quality monitoring: lessons from a California demonstration project. Marine Technology Society Journal, 45 (2): 65-73.
[27] Harwood, V. J., Staley, C., Badgley, B. D., Borges, K. and Korajkic, A. (2014). Microbial source tracking markers for detection of fecal contamination in environmental waters: relationships between pathogens and human health outcomes. FEMS Microbiological Reviews, 38 (1): 1-40.
[28] Cao, Y., Sivaganesan, M., Kinzelman, J., Blackwood, A. D., Noble, R. T., Haugland, R. A., Griffith, J. F. and Weisberg, S. B. (2013). Effect of platform, reference material, and quantification model on enumeration of Enterococcus by quantitative PCR methods. Water Research, 47 (1): 233-241.
[29] Sivaganesan, M., Siefring, S., Varma, M., and Haugland, R. A. (2011). MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples. Journal of Microbiological Methods, 87 (3): 343-349.
[30] Joensson, H. N., and Adersson, S. H. (2012). Droplet microfluidics – A tool for single-cell analysis. Angewandte Chemie International Edition, 51 (3): 12176-12192.
[31] Floren, C., Wiedemann, I., Brenig, B., Schűtz, E., and Beck, J. (2015). Species identification and quantification in meat and meat products using droplet digital PCR (DDPCR). Food Chemistry, 173: 1054-1058.
[32] Eastburn, D. J., Huang, Y., Pellegrino, M., Sciambi, A., Ptáček, L. J., and Abate, A. R. (2015). Microfluidic droplet enrichment for targeted sequencing. Nucleic Acids Research, 43 (13): e86. http://dx.doi.org/10.1093/nar/gkv297.
[33] Scollo, F., Egea, L. A., Gentile, A., La Malfa, S., Dorado, G., and Hernandez, P. (2016). Absolute quantification of olive oil DNA by droplet digital-PCR (DDPCR): comparison of isolation and amplification methodologies. Food Chemistry, 213: 388-394.
[34] Memon, A. A., Zőller, B., Hedelius, A., Wang, X., Stenman, E., Sundquist, J., and Sundquist, K. (2017). Quantification of mitochondrial DNA copy number in suspected cancer patients by a well optimized DDPCR method. Biomolecular Detection and Quantification, 13: 32-39.
[35] Periyannan Rajeswari, P. K., Soderberg, L. M., Yacoub, A., Leijon, M., Andersson Svahn, H., and Joensson, H. N. (2017). Multiple pathogen biomarker detection using an encoded bead array in droplet PCR. Journal of Microbiological Methods, 129: 22-28.
[36] Horan, N. J. (2003). Handbook of water and wastewater microbiology: Chapter 7 – Fecal indicator organisms. Academic press, 105-112.
[37] International Organization for Standardization. ISO/TS 12869: 2012 [S/OL]. [2012-10-12]. https://www.iso.org/standard/52079.html.
[38] Cao, Y., Griffith, J. F., Dorevitch, S., and Weisberg, S. B. (2012). Effectiveness of a qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters. Journal of Applied Microbiology, 113 (1): 66-75.
[39] Dong, L. H., Meng, Y., Wang, J. and Liu, Y. Y. (2014). Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers. Analytical and Bioanalytical Chemistry, 406 (6): 1701-1712.
[40] Cao, Y. P., Raith, M. R., and Griffith, J. F. (2015). Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment. Water Research, 70: 337-349.
Author Information
  • Laboratory and Research Centre, The Macao Water Supply Company Limited, Macao SAR, China

  • Laboratory and Research Centre, The Macao Water Supply Company Limited, Macao SAR, China

  • Laboratory and Research Centre, The Macao Water Supply Company Limited, Macao SAR, China

  • Laboratory and Research Centre, The Macao Water Supply Company Limited, Macao SAR, China

  • Ecological Environment Institute, Chinese Academy of Environmental Planning, Beijing, China

  • Laboratory and Research Centre, The Macao Water Supply Company Limited, Macao SAR, China

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  • APA Style

    Wei Ma, Yi Jun Kong, Weng U Ho, Si Ian Lam, Gui Huan Liu, et al. (2020). The Development and Application of DDPCR Technology on Quantification of Total Coliforms in Water. American Journal of Environmental Protection, 9(2), 22-30. https://doi.org/10.11648/j.ajep.20200902.11

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    ACS Style

    Wei Ma; Yi Jun Kong; Weng U Ho; Si Ian Lam; Gui Huan Liu, et al. The Development and Application of DDPCR Technology on Quantification of Total Coliforms in Water. Am. J. Environ. Prot. 2020, 9(2), 22-30. doi: 10.11648/j.ajep.20200902.11

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    AMA Style

    Wei Ma, Yi Jun Kong, Weng U Ho, Si Ian Lam, Gui Huan Liu, et al. The Development and Application of DDPCR Technology on Quantification of Total Coliforms in Water. Am J Environ Prot. 2020;9(2):22-30. doi: 10.11648/j.ajep.20200902.11

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  • @article{10.11648/j.ajep.20200902.11,
      author = {Wei Ma and Yi Jun Kong and Weng U Ho and Si Ian Lam and Gui Huan Liu and Sin Neng Chio},
      title = {The Development and Application of DDPCR Technology on Quantification of Total Coliforms in Water},
      journal = {American Journal of Environmental Protection},
      volume = {9},
      number = {2},
      pages = {22-30},
      doi = {10.11648/j.ajep.20200902.11},
      url = {https://doi.org/10.11648/j.ajep.20200902.11},
      eprint = {https://download.sciencepg.com/pdf/10.11648.j.ajep.20200902.11},
      abstract = {In this research, the detection method for absolute quantification of total coliforms was established based on Droplet Digital Polymerase Chain Reaction (DDPCR) technology using lacZ as the target gene for coliform group detection. The experimental conditions (e.g. primer and probe concentrations, annealing temperatures, etc) were well optimized. Besides, the linear range, precision and limit of quantification (LOQ) of this method were investigated and evaluated. The results illustrated that the optimal primer concentration was 0.2 μmol/L, whereas the optimal probe concentration was 0.5 μmol/L. The optimal annealing temperature was 56°C. The linear relationship between the total coliform genome DNA concentrations derived from DDPCR and DNA fluorometer was quite good (R2 = 0.999). The linear range was 3.95 ~ 7.80 × 104 copies/20 μL DDPCR reaction system. The LOQ for total coliforms was single copy per reaction system. Practical applications using real water samples collected from water supply system in Macao illustrated that this innovative method possessed high efficiencies and capabilities. This is probably the first research using DDPCR technology to absolutely qualify and quantify total coliforms and successfully applied it in Macao water supply system. The achievements from this research could provide with significant values for setting-up the emergency mechanism of water pollution in early stage.},
     year = {2020}
    }
    

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  • TY  - JOUR
    T1  - The Development and Application of DDPCR Technology on Quantification of Total Coliforms in Water
    AU  - Wei Ma
    AU  - Yi Jun Kong
    AU  - Weng U Ho
    AU  - Si Ian Lam
    AU  - Gui Huan Liu
    AU  - Sin Neng Chio
    Y1  - 2020/05/14
    PY  - 2020
    N1  - https://doi.org/10.11648/j.ajep.20200902.11
    DO  - 10.11648/j.ajep.20200902.11
    T2  - American Journal of Environmental Protection
    JF  - American Journal of Environmental Protection
    JO  - American Journal of Environmental Protection
    SP  - 22
    EP  - 30
    PB  - Science Publishing Group
    SN  - 2328-5699
    UR  - https://doi.org/10.11648/j.ajep.20200902.11
    AB  - In this research, the detection method for absolute quantification of total coliforms was established based on Droplet Digital Polymerase Chain Reaction (DDPCR) technology using lacZ as the target gene for coliform group detection. The experimental conditions (e.g. primer and probe concentrations, annealing temperatures, etc) were well optimized. Besides, the linear range, precision and limit of quantification (LOQ) of this method were investigated and evaluated. The results illustrated that the optimal primer concentration was 0.2 μmol/L, whereas the optimal probe concentration was 0.5 μmol/L. The optimal annealing temperature was 56°C. The linear relationship between the total coliform genome DNA concentrations derived from DDPCR and DNA fluorometer was quite good (R2 = 0.999). The linear range was 3.95 ~ 7.80 × 104 copies/20 μL DDPCR reaction system. The LOQ for total coliforms was single copy per reaction system. Practical applications using real water samples collected from water supply system in Macao illustrated that this innovative method possessed high efficiencies and capabilities. This is probably the first research using DDPCR technology to absolutely qualify and quantify total coliforms and successfully applied it in Macao water supply system. The achievements from this research could provide with significant values for setting-up the emergency mechanism of water pollution in early stage.
    VL  - 9
    IS  - 2
    ER  - 

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