Evaluation of Molecular Methods for the Detection and Quantification of Pathogen-Derived Nucleic Acids in Sediment

Kata Farkas; Francis Hassard; James E. McDonald; Shelagh K. Malham; Davey L. Jones

doi:10.3389/fmicb.2017.00053

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Evaluation of Molecular Methods for the Detection and Quantification of Pathogen-Derived Nucleic Acids in Sediment

Journal article

Open access

Peer reviewed

Evaluation of Molecular Methods for the Detection and Quantification of Pathogen-Derived Nucleic Acids in Sediment

Kata Farkas, Francis Hassard, James E. McDonald, Shelagh K. Malham and Davey L. Jones

Frontiers in microbiology, Vol.8, 53

2017

DOI: https://doi.org/10.3389/fmicb.2017.00053

PMCID: PMC5258707

PMID: 28174565

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Abstract

dPCR

Microbiology

norovirus

nucleic acid extraction

pathogen detection

qRT-PCR

sediment

Vibrio

The accurate detection of pathogens in environmental matrices, such as sediment, is critical in understanding pathogen fate and behavior in the environment. In this study, we assessed the usefulness of methods for the detection and quantification of Vibrio spp. and norovirus (NoV) nucleic acids in sediment. For bacteria, a commonly used direct method using hexadecyltrimethylammonium bromide (CTAB) and phenol-chloroform-isoamyl alcohol (PCI) extraction was optimized, whereas for NoV, direct and indirect (virus elution—concentration) methods were evaluated. For quantification, commercially available quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) kits were tested alongside a digital PCR (dPCR) approach. CTAB-based extraction combined with 16 h polyethylene glycol 6000 (PEG6000) precipitation was found to be suitable for the direct extraction of high abundance bacterial and viral nucleic acids. For the indirect extraction of viral RNA, beef extract-based elution followed by PEG6000 precipitation and extraction using the NucliSENS® MiniMag® Nucleic Acid Purification System and the PowerViral® Environmental RNA/DNA Isolation Kit and qRT-PCR resulted in 83–112 and 63–69% recoveries of NoV, respectively. dPCR resulted in lower viral recoveries (47 and 9%) and ~4 orders of magnitude lower Vibrio concentrations (3.6–4.6 log10 gc/100 g sediment) than was observed using qPCR. The use of internal controls during viral quantification revealed that the RT step was more affected by inhibitors than the amplification. The methods described here are suitable for the enumeration of viral and/or bacterial pathogens in sediment, however the use of internal controls to assess efficiency is recommended.

Details

Title: Evaluation of Molecular Methods for the Detection and Quantification of Pathogen-Derived Nucleic Acids in Sediment
Authors/Creators: Kata Farkas - Bangor University
Francis Hassard - Bangor University
James E. McDonald - Bangor University
Shelagh K. Malham - Bangor University
Davey L. Jones - Bangor University
Publication Details: Frontiers in microbiology, Vol.8, 53
Publisher: Frontiers Media S.A
Grant note: Food Standards Agency NE/M010996/1 / Natural Environment Research Council
Identifiers: 991005560323107891
Murdoch Affiliation: Centre for Sustainable Farming Systems
Language: English
Resource Type: Journal article

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136 Times Cited - Web of Science

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Collaboration types: Domestic collaboration
Citation topics: 1 Clinical & Life Sciences; 1.246 Diarrheal Diseases; 1.246.710 Enteric Viruses
Web Of Science research areas: Microbiology
ESI research areas: Microbiology