title: Evaluation of the celite secondary concentration procedure and an alternate elution buffer for the recovery of enteric adenoviruses 40 and 41.
abstract: The effective recovery of adenovirus from water is a critical first step in developing a virus occurrence method able to provide accurate data for risk assessments and other applications. During virus concentration, electropositive filters are typically eluted with beef extract, undergo secondary concentration using either an organic flocculation or polyethylene glycol (PEG) precipitation technique and are ultimately resuspended in sodium phosphate buffer. In this study, an alternative secondary concentration procedure using celite was optimized by identifying the optimal celite and elution buffer to use. Two elution buffers, sodium phosphate and 1× PBS, were evaluated for their impact on real-time PCR. Sodium phosphate produced high levels of PCR inhibition compared to 1× PBS and so 1× PBS was used in subsequent experiments. The two secondary concentration techniques that were tested with adenovirus 40 and 41 gave recoveries of 69% and 65% for the optimized celite method and 75% and 109% for the organic flocculation method, respectively. Fine particle, calcinated celites in combination with 1× PBS elution buffer were shown to be effective at concentrating adenovirus 40 and 41 during secondary concentration and their subsequent detection using PCR. Heat extraction efficiencies were compared to samples processed using a DNA extraction kit to address possible virus aggregation issues. Samples processed through DNA extraction were found to produce realistic adenovirus recoveries compared to exaggerated recoveries using heat extraction.
authors.affiliation: Biohazard Assessment Research Branch, Microbial and Chemical Exposure Assessment Research Division, National Exposure Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Cincinnati, OH 45268, USA. mcminn.brian@epa.gov
authors.forename: Brian R
authors.initials: BR
authors.lastname: McMinn
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Cashdollar
authors.forename: Ann C
authors.initials: AC
authors.lastname: Grimm
authors.forename: G Shay
authors.initials: GS
authors.lastname: Fout
mesh_terms: D000256:Adenoviridae / Q000302:isolation & purification*; D000818:Animals; D002021:Buffers; D007692:Diatomaceous Earth; D006801:Humans; D013048:Specimen Handling / Q000379:methods*; D014773:Virology / Q000379:methods*; D014871:Water Microbiology
chemical_list: D002021:Buffers; D007692:Diatomaceous Earth