Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens

A universal filtration and enzyme-based workflow has been established to allow for the rapid and sensitive quantification of leading pathogens <i>Cryptosporidium parvum, Giardia gamblia, Campylobacter jejuni,</i> and <i>Escherichia coli</i> from tap water samples with volumes...

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Autores principales:	Angela Sun, Jo-Ann L. Stanton, Peter L. Bergquist, Anwar Sunna
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	qPCR quantification waterborne pathogen enumeration filtration potable water <i>Campylobacter jejuni</i> Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/e8c5e29b0c0f499dbfe1805a3cb5b180
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spelling	oai:doaj.org-article:e8c5e29b0c0f499dbfe1805a3cb5b1802021-11-25T18:25:28ZUniversal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens10.3390/microorganisms91123672076-2607https://doaj.org/article/e8c5e29b0c0f499dbfe1805a3cb5b1802021-11-01T00:00:00Zhttps://www.mdpi.com/2076-2607/9/11/2367https://doaj.org/toc/2076-2607A universal filtration and enzyme-based workflow has been established to allow for the rapid and sensitive quantification of leading pathogens <i>Cryptosporidium parvum, Giardia gamblia, Campylobacter jejuni,</i> and <i>Escherichia coli</i> from tap water samples with volumes up to 100 mL, and the potential to scale up to larger volumes. qPCR limits of quantification as low as four oocysts for <i>Cryptosporidium</i>, twelve cysts for <i>Giardia</i>, two cells for <i>C. jejuni</i>, and nineteen cells for <i>E. coli</i> per reaction were achieved. A polycarbonate filter-based sampling method coupled with the prepGEM enzyme-based DNA extraction system created a single-step transfer workflow that required as little as 20 min of incubation time and a 100 µL reaction mix. The quantification via qPCR was performed directly on the prepGEM extract, bypassing time-consuming, labour-intensive conventional culture-based methods. The tap water samples were shown to contain insoluble particles that inhibited detection by reducing the quantification efficiency of a representative pathogen (<i>C. jejuni</i>) to 30–60%. This sample inhibition was effectively removed by an on-filter treatment of 20% (<i>v</i>/<i>v</i>) phosphoric acid wash. Overall, the established workflow was able to achieve quantification efficiencies of 92% and higher for all four leading water pathogens, forming the basis of a rapid, portable, and low-cost solution to water monitoring.Angela SunJo-Ann L. StantonPeter L. BergquistAnwar SunnaMDPI AGarticleqPCR quantificationwaterborne pathogenenumerationfiltrationpotable water<i>Campylobacter jejuni</i>Biology (General)QH301-705.5ENMicroorganisms, Vol 9, Iss 2367, p 2367 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	qPCR quantification waterborne pathogen enumeration filtration potable water <i>Campylobacter jejuni</i> Biology (General) QH301-705.5
spellingShingle	qPCR quantification waterborne pathogen enumeration filtration potable water <i>Campylobacter jejuni</i> Biology (General) QH301-705.5 Angela Sun Jo-Ann L. Stanton Peter L. Bergquist Anwar Sunna Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens
description	A universal filtration and enzyme-based workflow has been established to allow for the rapid and sensitive quantification of leading pathogens <i>Cryptosporidium parvum, Giardia gamblia, Campylobacter jejuni,</i> and <i>Escherichia coli</i> from tap water samples with volumes up to 100 mL, and the potential to scale up to larger volumes. qPCR limits of quantification as low as four oocysts for <i>Cryptosporidium</i>, twelve cysts for <i>Giardia</i>, two cells for <i>C. jejuni</i>, and nineteen cells for <i>E. coli</i> per reaction were achieved. A polycarbonate filter-based sampling method coupled with the prepGEM enzyme-based DNA extraction system created a single-step transfer workflow that required as little as 20 min of incubation time and a 100 µL reaction mix. The quantification via qPCR was performed directly on the prepGEM extract, bypassing time-consuming, labour-intensive conventional culture-based methods. The tap water samples were shown to contain insoluble particles that inhibited detection by reducing the quantification efficiency of a representative pathogen (<i>C. jejuni</i>) to 30–60%. This sample inhibition was effectively removed by an on-filter treatment of 20% (<i>v</i>/<i>v</i>) phosphoric acid wash. Overall, the established workflow was able to achieve quantification efficiencies of 92% and higher for all four leading water pathogens, forming the basis of a rapid, portable, and low-cost solution to water monitoring.
format	article
author	Angela Sun Jo-Ann L. Stanton Peter L. Bergquist Anwar Sunna
author_facet	Angela Sun Jo-Ann L. Stanton Peter L. Bergquist Anwar Sunna
author_sort	Angela Sun
title	Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens
title_short	Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens
title_full	Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens
title_fullStr	Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens
title_full_unstemmed	Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens
title_sort	universal enzyme-based field workflow for rapid and sensitive quantification of water pathogens
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/e8c5e29b0c0f499dbfe1805a3cb5b180
work_keys_str_mv	AT angelasun universalenzymebasedfieldworkflowforrapidandsensitivequantificationofwaterpathogens AT joannlstanton universalenzymebasedfieldworkflowforrapidandsensitivequantificationofwaterpathogens AT peterlbergquist universalenzymebasedfieldworkflowforrapidandsensitivequantificationofwaterpathogens AT anwarsunna universalenzymebasedfieldworkflowforrapidandsensitivequantificationofwaterpathogens
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Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens

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