Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test
Abstract Over the last decades, various PCR-based methods have been proposed that can identify sources of faecal pollution in environmental waters. These microbial source tracking (MST) methods are powerful tools to manage water quality and support public health risk assessment. However, their appli...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2019-01-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-018-36749-7 |
| _version_ | 1818995996856680448 |
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| author | Claudia Kolm Roland Martzy Manuela Führer Robert L. Mach Rudolf Krska Sabine Baumgartner Andreas H. Farnleitner Georg H. Reischer |
| author_facet | Claudia Kolm Roland Martzy Manuela Führer Robert L. Mach Rudolf Krska Sabine Baumgartner Andreas H. Farnleitner Georg H. Reischer |
| author_sort | Claudia Kolm |
| collection | DOAJ |
| description | Abstract Over the last decades, various PCR-based methods have been proposed that can identify sources of faecal pollution in environmental waters. These microbial source tracking (MST) methods are powerful tools to manage water quality and support public health risk assessment. However, their application is limited by the lack of specialized equipment and trained personnel in laboratories performing microbiological water quality assessment. Here, we describe a novel molecular method that combines helicase-dependent amplification (HDA) with a strip test for detecting ruminant faecal pollution sources. Unlike quantitative PCR (qPCR), the developed HDA-strip assay only requires a heating block to amplify the ruminant-associated Bacteroidetes 16S rRNA marker (BacR). Following HDA, the reaction mixture can be directly applied onto the test strip, which detects and displays the amplification products by marker-specific hybridization probes via an on-strip colorimetric reaction. The entire assay takes two hours and demands no extensive practical training. Furthermore, the BacR HDA-strip assay achieved comparable results in head-to-head performance tests with the qPCR reference, in which we investigated source-sensitivity and source-specificity, the analytical limit of detection, and the sample limit of detection. Although this approach only yields qualitative results, it can pave a way for future simple-to-use MST screening tools. |
| first_indexed | 2024-12-20T21:22:43Z |
| format | Article |
| id | doaj.art-456c3a09e3824df08b57dbaab6349f5c |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-20T21:22:43Z |
| publishDate | 2019-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-456c3a09e3824df08b57dbaab6349f5c2022-12-21T19:26:13ZengNature PortfolioScientific Reports2045-23222019-01-01911910.1038/s41598-018-36749-7Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip testClaudia Kolm0Roland Martzy1Manuela Führer2Robert L. Mach3Rudolf Krska4Sabine Baumgartner5Andreas H. Farnleitner6Georg H. Reischer7TU Wien, Institute of Chemical, Environmental & Bioscience Engineering, Molecular Diagnostics Group, Department IFA-TullnTU Wien, Institute of Chemical, Environmental & Bioscience Engineering, Molecular Diagnostics Group, Department IFA-TullnUniversity of Natural Resources and Life Sciences, Vienna (BOKU), Department IFA-Tulln, Center for Analytical ChemistryTU Wien, Institute of Chemical, Environmental & Bioscience Engineering, Research Division Biochemical Technology, Research Group Synthetic Biology and Molecular BiotechnologyUniversity of Natural Resources and Life Sciences, Vienna (BOKU), Department IFA-Tulln, Center for Analytical ChemistryUniversity of Natural Resources and Life Sciences, Vienna (BOKU), Department IFA-Tulln, Center for Analytical ChemistryICC Interuniversity Cooperation Centre Water & HealthTU Wien, Institute of Chemical, Environmental & Bioscience Engineering, Molecular Diagnostics Group, Department IFA-TullnAbstract Over the last decades, various PCR-based methods have been proposed that can identify sources of faecal pollution in environmental waters. These microbial source tracking (MST) methods are powerful tools to manage water quality and support public health risk assessment. However, their application is limited by the lack of specialized equipment and trained personnel in laboratories performing microbiological water quality assessment. Here, we describe a novel molecular method that combines helicase-dependent amplification (HDA) with a strip test for detecting ruminant faecal pollution sources. Unlike quantitative PCR (qPCR), the developed HDA-strip assay only requires a heating block to amplify the ruminant-associated Bacteroidetes 16S rRNA marker (BacR). Following HDA, the reaction mixture can be directly applied onto the test strip, which detects and displays the amplification products by marker-specific hybridization probes via an on-strip colorimetric reaction. The entire assay takes two hours and demands no extensive practical training. Furthermore, the BacR HDA-strip assay achieved comparable results in head-to-head performance tests with the qPCR reference, in which we investigated source-sensitivity and source-specificity, the analytical limit of detection, and the sample limit of detection. Although this approach only yields qualitative results, it can pave a way for future simple-to-use MST screening tools.https://doi.org/10.1038/s41598-018-36749-7 |
| spellingShingle | Claudia Kolm Roland Martzy Manuela Führer Robert L. Mach Rudolf Krska Sabine Baumgartner Andreas H. Farnleitner Georg H. Reischer Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test Scientific Reports |
| title | Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test |
| title_full | Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test |
| title_fullStr | Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test |
| title_full_unstemmed | Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test |
| title_short | Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test |
| title_sort | detection of a microbial source tracking marker by isothermal helicase dependent amplification and a nucleic acid lateral flow strip test |
| url | https://doi.org/10.1038/s41598-018-36749-7 |
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