Rapid quantification of total viral DNA in the supernatants of activated sludge samples with the fluorescent dye PicoGreen®

Abstract

To develop a rapid and simple method for quantifying viral DNA concentrations and determining viral quantities in activated sludge. Activated sludge samples were obtained from three full-scale and one laboratory-scale process. They were centrifuged and the supernatant was filtered through a 0.2-microm membrane filter. Free DNA was removed by DNase-I treatment; any DNA within the viral capsid was liberated by heat treatment and proteinase K, and viral DNA concentrations were determined using the dye PicoGreen. To validate the method, we assessed the recovery of T4 phage added to filtered samples, which was 99% of those added. Viral DNA concentrations in samples from full-scale plants ranged from 69 to 157 ng ml(-1). Monitoring of laboratory-scale reactor samples revealed that viral DNA concentrations varied with time. Our method involves a simple sample treatment protocol and allow rapid analysis of many samples. A simple, rapid and sensitive method was developed and successfully used to determine the viral DNA concentrations in activated sludge. This method provides a way to investigate impact of bacteriophages on the performance of wastewater treatment processes.