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Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters - PubMed

Comparative Study

Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters

W Ahmed et al. Appl Environ Microbiol. 2015 Mar.

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 on HA membranes (90 mm in 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 with NaOH and 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 (Mg(2+)) 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.

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Figures

FIG 1
FIG 1

Procedures for virus concentration methods in sewage spiked tap and river water samples. Method A is direct nucleic acid extraction from negatively charged membranes, method B includes an adsorption/elution-based protocols with negatively charged membranes, and method C is a modified version of adsorption/elution-based protocols with negatively charged membranes.

FIG 2
FIG 2

Box-and-whisker plots of the concentration of gene copies of HAdVs (a) and HPyVs (b) in sewage spiked tap and river water samples. The inner box lines represent the medians, while the outer box lines represent the 25th and 75th percentiles.

FIG 3
FIG 3

Box-and-whisker plots of the concentration of gene copies of HAdVs (a) and HPyVs (b) in nucleic acid samples extracted from eluates and membranes using method B. The inner box lines represent the medians, while the outer box lines represent the 25th and 75th percentiles.

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References

    1. Ahmed W, Neller R, Katouli M. 2005. Evidence of septic system failure determined by a bacterial biochemical fingerprinting method. J Appl Microbiol 98:910–920. doi:10.1111/j.1365-2672.2004.02522.x. - DOI - PubMed
    1. Byappanahalli MN, Nevers MB, Korajkic A, Staley ZR, Harwood VJ. 2012. Enterococci in the environment. Microbiol Mol Biol Rev 76:685–706. doi:10.1128/MMBR.00023-12. - DOI - PMC - PubMed
    1. Harwood VJ, Staley C, Badgley BD, Borges K, Korajkic A. 2014. Microbial source tracking markers for detection of fecal contamination in environmental waters: relationships between pathogens and human health outcomes. FEMS Microbiol Rev 38:1–40. doi:10.1111/1574-6976.12031. - DOI - PubMed
    1. McQuaig S, Griffith J, Harwood VJ. 2012. Association of fecal indicator bacteria with human viruses and microbial source tracking markers at coastal beaches impacted by nonpoint source pollution. Appl Environ Microbiol 78:6423–6432. doi:10.1128/AEM.00024-12. - DOI - PMC - PubMed
    1. Sidhu JPS, Hodgers L, Ahmed W, Chong MN, Toze S. 2012. Prevalence of human pathogens and indicators in stormwater runoff in Brisbane, Australia. Water Res 46:6652–6660. doi:10.1016/j.watres.2012.03.012. - DOI - PubMed

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