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

Water samples, in a non-cytotoxic dilution, were inoculated in triplicate in A549 cells for the ICC-RT-qPCR assay. After 1 h of incubation at 37°C with rotation every 15 min, the inoculum was removed and the cell layers were overlaid with high-glucose Dulbecco's Modified Eagle's Medium (DMEM) before being incubated at 37°C for 24 h. After incubation as described previously, the supernatant was recovered and 0.2 mL was used for genetic material extraction, as described above. Immediately after the extraction of the total nucleic acids, enzymatic treatment, with DNase I, was conducted in order to degrade the DNA in the sample tested. Then a reverse transcriptase reaction (RT) was used to generate cDNA from viral mRNA. The quantification of infectious particles of HAdV was performed with qPCR, as described above.