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...

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Autores principales:	Claudia Kolm, Roland Martzy, Manuela Führer, Robert L. Mach, Rudolf Krska, Sabine Baumgartner, Andreas H. Farnleitner, Georg H. Reischer
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2019
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/456c3a09e3824df08b57dbaab6349f5c
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spelling	oai:doaj.org-article:456c3a09e3824df08b57dbaab6349f5c2021-12-02T15:08:09ZDetection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test10.1038/s41598-018-36749-72045-2322https://doaj.org/article/456c3a09e3824df08b57dbaab6349f5c2019-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-36749-7https://doaj.org/toc/2045-2322Abstract 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.Claudia KolmRoland MartzyManuela FührerRobert L. MachRudolf KrskaSabine BaumgartnerAndreas H. FarnleitnerGeorg H. ReischerNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 9, Iss 1, Pp 1-9 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q 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
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.
format	article
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
title	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_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_sort	detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test
publisher	Nature Portfolio
publishDate	2019
url	https://doaj.org/article/456c3a09e3824df08b57dbaab6349f5c
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Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test

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