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Resin-based method for concentration of enteric viruses and F-RNA coliphages from water samples




Perez-Méndez, Alma Topiltzin, author
Goodridge, Lawrence, advisor
Marshall, Douglas, committee member
Nightingale, Kendra, committee member
Van Campen, Hana, committee member

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Fecal contamination of source and recreational waters represents a public health concern due to potential content of human pathogens, and the variety of sources from which an individual may be exposed to such contamination. Enteric viruses such as noroviruses, rotaviruses, adenoviruses and hepatitis viruses are dispersed by fecal contamination and are a major cause of waterborne diseases in the US and worldwide. Given the variety of viral enteric pathogens and their particular growth requirements, their detection is technically difficult and time consuming. An alternative to determine the risk of enteric virus contamination in water is to detect viral indicators of fecal contamination. F-RNA coliphages are recognized as enteric virus surrogates, fecal indicators useful for source tracking. Enteric viruses and F-RNA coliphages are often present at low concentrations in contaminated waters; therefore rapid, sensitive and cost effective viral concentration methods applicable to different environmental water samples are needed for an accurate assessment of water microbiological safety. Here, a resin-based virus concentration method was developed and tested. The method is based on adsorption of the viruses to an anion exchange resin dispersed in the water sample, followed by direct isolation of nucleic acids from the resin to provide a small volume final sample. In order to test the method with a wide variety of viral structures and characteristics, three enteric viruses (hepatitis A virus, adenovirus and rotavirus) and four F-RNA coliphages were used. Additionally, tap water and a variety of environmental samples were tested. After virus concentration, detection was performed through real time RT-PCR, a sensitive molecular technique widely use for detection of these viruses. In tap water containing 105 pfu/ml of F-RNA coliphages, the anion exchange resin adsorbed over 96% of the coliphage present, allowing for detection of between 100 to 10-1 pfu/ml of F-RNA coliphages in 50 ml samples. Similarly, experiments with large volumes of tap water showed that the resin-based method was capable of detection limits as low as 10 TCID50 of enteric viruses in tap water. Finally, the evaluation of the method with different samples of environmental water showed that the resin was useful for concentration of F-RNA coliphages in most of the samples, despite the presence of PCR inhibitors in the water. Limitations of the method included incomplete recovery of nucleic acids from the resin and concentration of PCR inhibitors from the samples. Given the simplicity of the method and the promising results obtained in this work, studies focused in increasing the yield of nucleic acids and decreasing the concentration of environmental inhibitors in the concentrated sample is warranted.


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anion exchange resin
F-RNA bacteriophages
source tracking
virus concentration methods
enteric viruses
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