Evaluation and molecular characterization of human adenovirus in drinking water supplies: viral integrity and viability assays

Fongaro, Gislaine; Nascimento, Mariana A do; Rigotto, Caroline; Ritterbusch, Giseli; da Silva, Alessandra D’ A; Esteves, Paulo A; Barardi, Célia R M

To infer the presence of undamaged viral particles, HAdV-positive samples, as detected by qPCR, were treated with DNase I, as described by Viancelli et al. (2011) [42] . To verify potential inhibitors of DNAse I present in the sample matrix, a known amount of previously inactivated HAdV-2 (1 h at 99°C and 30 min under UV irradiation) was added in concentrated samples (previously HAdV-2 negative) of all sites and in nuclease-free water (NFW), as a control. The reactions were performed using 1 U of DNAse (sufficient quantity to degrade 100% of DNA added), 1× buffer and 180 μL of sample or NFW and incubated for 15 min at room temperature, with the intention of degrading all free genetic material. Then, the enzyme was inactivated with EDTA 25 mM and incubated for 10 minutes at 65°C. These treated samples/NFW were then subjected to nucleic acid extraction and qPCR, as described previously.