Three-step isolation method for sensitive detection of enterovirus, rotavirus, hepatitis A virus, and small round...

Control of drinking or bathing water quality in respect to viral contamination remains an unsolved problem. A highly sensitive isolation protocol was developed for concentration and detection of different enteric viruses from water samples. The three-step isolation procedure combines filtration with a positively charged nylon membrane, ultrafiltration and clean-up of the viral RNA with a silica based membrane. Detection of the viral RNA is accomplished by reverse-transcription polymerase chain reaction (RT-PCR). Detection limits were determined to be one 50% tissue culture infective dose (TCID50) of seeded coxsackievirus B2 or hepatitis A Virus per litre of tap water by RT-PCR compared to two orders of magnitude lower sensitivity for culture in the case of coxsackievirus B2. The isolation procedure is highly sensitive, easy to perform and allows the detection of different human pathogenic virus groups in one water sample. The application of the isolation procedure to six river water samples and subsequent detection with nested or semi-nested PCR revealed enterovirus in 6/6 (100%), rotavirus in 6/6 (100%), hepatitis A virus in 0/6 (0%), small round structured virus genotype I in 6/6 (100%) and small round structured virus genotype II in 2/6 (33%) of the samples. These findings suggest that first, we have developed a very sensitive detection procedure and second, that river water in Switzerland-where most of the wastewater is handled by sewage treatment plants-shows a high contamination rate with enteric viruses.