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.
| Published in | American Journal of Environmental Protection (Volume 9, Issue 2) |
| DOI | 10.11648/j.ajep.20200902.11 |
| 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), 2020. Published by Science Publishing Group |
Total Coliforms, DDPCR, Absolute Quantification, Water Supply, Water-borne Pathogen
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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
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
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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Download@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://article.sciencepublishinggroup.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}
}
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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