Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test

Claudia Kolm,Andreas H Farnleitner,Rudolf Krska,Roland Martzy,Manuela Führer,Georg H Reischer,Sabine Baumgartner,Robert L Mach

doi:10.1038/s41598-018-36749-7

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.

Highlights

Faecal pollution of water is a serious problem that affects water systems in both developing and industrialized countries
The method relies on an asymmetric helicase-dependent amplification (HDA) assay that is entirely performed on a standard heating block and that produces single-stranded Bacteroidetes 16S rRNA marker (BacR) copies once the limiting primer is used up (Fig. 1a, Step 1)
We developed the first HDA-strip assay for identifying faecal pollution sources in water by targeting one of the most prominent microbial source tracking markers, BacR

Summary

Introduction

Faecal pollution of water is a serious problem that affects water systems in both developing and industrialized countries. The PCR-like reaction scheme of HDA (i.e., the use of two primers to flank the genetic marker) allows for the adaption of PCR assays to the HDA system[15,16] This makes HDA an appealing method for settings with limited resources and infrastructure, especially when combined with paper-based analytical devices that enable rapid, simple and low-cost detection of amplification products[15,17]. To provide a foundation for future, rapid, simple-to-use and low-cost molecular source tracking tools, we developed an HDA-strip assay to detect the ruminant-associated genetic marker (16S rRNA) in faecal members of the phylum Bacteroides This marker – referred to as “BacR” – is traditionally determined by qPCR27 and has become one of the most widely used MST assays to detect ruminant faecal pollution sources in environmental waters. The specific aims were (i) to both design and develop a BacR HDA assay and a BacR strip test and (ii) to assess their combined performance in comparison to the qPCR reference method in experiments investigating source-sensitivity, source-specificity, the analytical limit of detection, and the sample limit of detection

Methods

Results

Conclusion