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: | , , , |
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| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
MDPI AG
2021
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/e8c5e29b0c0f499dbfe1805a3cb5b180 |
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| Sumario: | 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. |
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