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A highly specific Escherichia coli qPCR and its comparison with existing methods for environmental waters

A highly specific Escherichia coli qPCR and its comparison with existing methods for environmental waters
A highly specific Escherichia coli qPCR and its comparison with existing methods for environmental waters
The presence of Escherichia coli in environmental waters is considered as evidence of faecal contamination and is therefore commonly used as an indicator in both water quality and food safety analysis. The long period of time between sample collection and obtaining results from existing culture based methods means that contamination events may already impact public health by the time they are detected. The adoption of molecular based methods for E. coli could significantly reduce the time to detection. A new quantitative real-time PCR (qPCR) assay was developed to detect the ybbW gene sequence, which was found to be 100% exclusive and inclusive (specific and sensitive) for E. coli and directly compared for its ability to quantify E. coli in environmental waters against colony counts, quantitative real-time NASBA (qNASBA) targeting clpB and qPCR targeting uidA. Of the 87 E. coli strains tested, 100% were found to be ybbW positive, 94.2% were culture positive, 100% were clpB positive and 98.9% were uidA positive. The qPCR assays had a linear range of quantification over several orders of magnitude, and had high amplification efficiencies when using single isolates as a template. This compared favourably with qNASBA which showed poor linearity and amplification efficiency. When the assays were applied to environmental water samples, qNASBA was unable to reliably quantify E. coli while both qPCR assays were capable of predicting E. coli concentrations in environmental waters. This study highlights the inability of qNASBA targeting mRNA to quantify E. coli in environmental waters, and presents the first E. coli qPCR assay with 100% target exclusivity. The application of a highly exclusive and inclusive qPCR assay has the potential to allow water quality managers to reliably and rapidly detect and quantify E. coli and therefore take appropriate measures to reduce the risk to public health posed by faecal contamination.
0043-1354
101-110
Walker, David I.
17d6709b-4e0c-4f72-9a9e-78c177ab4542
McQuillan, Jonathan S.
697cdf72-f353-4779-b64a-45494f29772f
Taiwo, Michael
3ac7811b-4d5d-487b-9a49-4453595c2e61
Parks, Rachel
342f6345-2cbb-4480-921f-97e5d8a6265d
Stenton, Craig A.
e7880000-c3b9-4c7d-8e67-26483e5386f9
Morgan, Hywel
de00d59f-a5a2-48c4-a99a-1d5dd7854174
Mowlem, Matthew C.
6f633ca2-298f-48ee-a025-ce52dd62124f
Lees, David N.
276df848-c299-43bb-a340-43ebd8604541
Walker, David I.
17d6709b-4e0c-4f72-9a9e-78c177ab4542
McQuillan, Jonathan S.
697cdf72-f353-4779-b64a-45494f29772f
Taiwo, Michael
3ac7811b-4d5d-487b-9a49-4453595c2e61
Parks, Rachel
342f6345-2cbb-4480-921f-97e5d8a6265d
Stenton, Craig A.
e7880000-c3b9-4c7d-8e67-26483e5386f9
Morgan, Hywel
de00d59f-a5a2-48c4-a99a-1d5dd7854174
Mowlem, Matthew C.
6f633ca2-298f-48ee-a025-ce52dd62124f
Lees, David N.
276df848-c299-43bb-a340-43ebd8604541

Walker, David I., McQuillan, Jonathan S., Taiwo, Michael, Parks, Rachel, Stenton, Craig A., Morgan, Hywel, Mowlem, Matthew C. and Lees, David N. (2017) A highly specific Escherichia coli qPCR and its comparison with existing methods for environmental waters. Water Research, 126, 101-110. (doi:10.1016/j.watres.2017.08.032).

Record type: Article

Abstract

The presence of Escherichia coli in environmental waters is considered as evidence of faecal contamination and is therefore commonly used as an indicator in both water quality and food safety analysis. The long period of time between sample collection and obtaining results from existing culture based methods means that contamination events may already impact public health by the time they are detected. The adoption of molecular based methods for E. coli could significantly reduce the time to detection. A new quantitative real-time PCR (qPCR) assay was developed to detect the ybbW gene sequence, which was found to be 100% exclusive and inclusive (specific and sensitive) for E. coli and directly compared for its ability to quantify E. coli in environmental waters against colony counts, quantitative real-time NASBA (qNASBA) targeting clpB and qPCR targeting uidA. Of the 87 E. coli strains tested, 100% were found to be ybbW positive, 94.2% were culture positive, 100% were clpB positive and 98.9% were uidA positive. The qPCR assays had a linear range of quantification over several orders of magnitude, and had high amplification efficiencies when using single isolates as a template. This compared favourably with qNASBA which showed poor linearity and amplification efficiency. When the assays were applied to environmental water samples, qNASBA was unable to reliably quantify E. coli while both qPCR assays were capable of predicting E. coli concentrations in environmental waters. This study highlights the inability of qNASBA targeting mRNA to quantify E. coli in environmental waters, and presents the first E. coli qPCR assay with 100% target exclusivity. The application of a highly exclusive and inclusive qPCR assay has the potential to allow water quality managers to reliably and rapidly detect and quantify E. coli and therefore take appropriate measures to reduce the risk to public health posed by faecal contamination.

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Accepted/In Press date: 14 August 2017
e-pub ahead of print date: 25 August 2017
Published date: 1 December 2017

Identifiers

Local EPrints ID: 414496
URI: https://eprints.soton.ac.uk/id/eprint/414496
ISSN: 0043-1354
PURE UUID: 0b770d0e-3ece-461d-b7a8-aad1517b27f9
ORCID for Hywel Morgan: ORCID iD orcid.org/0000-0003-4850-5676

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Date deposited: 03 Oct 2017 16:31
Last modified: 10 Sep 2019 00:45

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