Application of PCR to the Detection of Adenoviruses in Polluted Waters

Abstract

A method for the detection of adenovirus in environmental samples has been developed. We tested several systems for concentrating viral particles by adding adenoviruses 2 and 12 to different sewage samples. The method selected was as follows: centrifugation of the sample in order to pellet adenovirus viral particles and all suspended solids, elution of the pelleted viruses by treatment with 0.25N glycine buffer pH 9.5, removal of solids from the sample by a short centrifugation and ultracentrifugation of the resulting supernatant. Elution with glycine buffer avoided inhibitors and showed more sensitivity than ultrasonication or filtration through a low binding protein filter to retain bacteria and suspended solids. Sewage samples were treated by this selected method and recovered viral particles were analyzed both by a two-step DNA amplification reaction and by infecting Hep-2 cells. About 50% of the samples were positive in a two-step PCR and these data were confirmed by tissue culture amplification and one step PCR. Two pairs of primers (external and nested) from the hexon region were used, which are able to detect human adenovirus from all subgenera. Although more studies are needed, the two-step PCR developed appears to be a quick and reliable method for adenovirus detection in environmental samples.