Detection of adenovirus and enterovirus by PCR amplification in polluted waters

Abstract

Several systems for virus recovery from environmental samples and extraction of nucleic acid were tested by adding adenovirus 2 and poliovirus 1 to different sewage samples. The most promising method involved: concentration of viruses by centrifugation, and elution of the pelleted viruses by treatment with 0.25 N glycine buffer pH 9.5. The nucleic acids were extracted by adsorption of RNA and DNA to silica particles. One aliquot was directly used for a two-step PCR in a nested fashion, with specific primers for all adenoviruses; the other aliquot was used to synthesize cDNA and a nested two-step PCR with specific primers for enteroviruses. The specificity and sensitivity of the selected primers were evaluated, the 47 human adenovirus serotypes were identified and 24 different enterovirus strains were recognized. The sensitivity of the nucleic acid extraction, cDNA synthesis and nested PCR amplification was estimated to be between 1 and 10 viral particles. Sewage and polluted river samples were analyzed showing, as expected, a much higher number of positive samples by the method described than by tissue culture analysis, a high prevalence of hepatitis A virus in sewage and the adenoviruses as the most commonly detected virus in the environmental samples analyzed.