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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-11-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/9/11/2367 |
| _version_ | 1827675973742493696 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T05:15:15Z |
| format | Article |
| id | doaj.art-e8c5e29b0c0f499dbfe1805a3cb5b180 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-10T05:15:15Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-e8c5e29b0c0f499dbfe1805a3cb5b1802023-11-23T00:30:24ZengMDPI AGMicroorganisms2076-26072021-11-01911236710.3390/microorganisms9112367Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water PathogensAngela Sun0Jo-Ann L. Stanton1Peter L. Bergquist2Anwar Sunna3Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, AustraliaDepartment of Anatomy, School of Biomedical Sciences, University of Otago, Dunedin 9054, New ZealandDepartment of Molecular Sciences, Macquarie University, Sydney, NSW 2109, AustraliaDepartment of Molecular Sciences, Macquarie University, Sydney, NSW 2109, AustraliaA 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.https://www.mdpi.com/2076-2607/9/11/2367qPCR quantificationwaterborne pathogenenumerationfiltrationpotable water<i>Campylobacter jejuni</i> |
| spellingShingle | Angela Sun Jo-Ann L. Stanton Peter L. Bergquist Anwar Sunna Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens Microorganisms qPCR quantification waterborne pathogen enumeration filtration potable water <i>Campylobacter jejuni</i> |
| title | 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_short | 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 |
| topic | qPCR quantification waterborne pathogen enumeration filtration potable water <i>Campylobacter jejuni</i> |
| url | https://www.mdpi.com/2076-2607/9/11/2367 |
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