Journal article
Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters
Applied and Environmental Microbiology, Vol.81(6), pp.2042-2049
2015
Abstract
Pathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. They are relatively difficult to concentrate from environmental waters due to typically low concentrations and their small size. Although rapid enumeration of viruses by quantitative PCR (qPCR) has the potential to greatly improve water quality analysis and risk assessment, the upstream steps of capturing and recovering viruses from environmental water sources along with removing PCR inhibitors from extracted nucleic acids remain formidable barriers to routine use. Here, we compared the efficiency of virus recovery for three rapid methods of concentrating two microbial source tracking (MST) viral markers human adenoviruses (HAdVs) and polyomaviruses (HPyVs) from one liter tap water and river water samples onHAmembranes (90mmin diameter). Samples were spiked with raw sewage, and viral adsorption to membranes was promoted by acidification (method A) or addition of MgCl2 (methods B and C). Viral nucleic acid was extracted directly from membranes (method A), or viruses were eluted withNaOHand concentrated by centrifugal ultrafiltration (methods B and C). No inhibition of qPCR was observed for samples processed by method A, but inhibition occurred in river samples processed by B and C. Recovery efficiencies of HAdVs and HPyVs were ~10-fold greater for method A (31 to 78%) than for methods B and C(2.4 to 12%). Further analysis of membranes from method B revealed that the majority of viruses were not eluted from the membrane, resulting in poor recovery. The modification of the originally published method A to include a larger diameter membrane and a nucleic acid extraction kit that could accommodate the membrane resulted in a rapid virus concentration method with good recovery and lack of inhibitory compounds. The frequently used strategy of viral absorption with added cations (Mg2+) and elution with acid were inefficient and more prone to inhibition, and will result in underestimation of the prevalence and concentrations of HAdVs and HPyVs markers in environmental waters.
Details
- Title
- Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters
- Authors
- Warish Ahmed (Author) - University of the Sunshine Coast - Faculty of Science, Health, Education and EngineeringV J Harwood (Author) - University of South Florida, United StatesP Gyawali (Author) - University of QueenslandJatinder P S Sidhu (Author) - University of the Sunshine Coast - Faculty of Science, Health, Education and EngineeringS Toze (Author) - University of Queensland
- Publication details
- Applied and Environmental Microbiology, Vol.81(6), pp.2042-2049
- Publisher
- American Society for Microbiology
- Date published
- 2015
- DOI
- 10.1128/AEM.03851-14
- ISSN
- 0099-2240
- Copyright note
- Copyright © 2015 American Society for Microbiology. The author's accepted version is reproduced here in accordance with the publisher's copyright policy. The definitive version is available at http://aem.asm.org/
- Organisation Unit
- University of the Sunshine Coast, Queensland
- Language
- English
- Record Identifier
- 99448935502621
- Output Type
- Journal article
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