A strategy for detection of viruses in groundwater by PCR

We evaluated the use of the PCR for detection of enteric viruses in groundw ater. To do this, we used an improved sample-processing technique and a lar ge-volume amplification protocol. The objective of this study was to use ad vanced molecular techniques to develop a rapid and simple method which can be used by the water industry for detection of viral contamination in a var iety of water samples. The strategy described here fulfills the water indus try's need for a rapid, reliable, easily performed method for analyzing gro undwater for virus contamination. Viruses were detected after concentration from at least 400 gallons (1,512 liters) of water by a filter adsorption a nd elution method, which resulted in a concentrate containing viruses. A to tal of 150 samples were analyzed by performing cell culture assays for ente roviruses and by performing reverse transcription PCR (RT-PCR) analyses for enteroviruses, hepatitis A virus, and rotavirus. Thirteen samples (8.7%) p roduced cellular cytopathic effects when the Buffalo green monkey cell line was used. When primers specific for enteroviruses were used in RT-PCR, 40 of 133 samples (30.1%) tested positive for the presence of enterovirus RNA. When hepatitis A virus-specific primers were used, 12 of 139 samples (8.6% ) were considered positive for the presence of hepatitis A viral RNA. The R T-PCR analysis performed with rotavirus-specific primers identified 18 of 1 30 samples (13.8%) that were positive for rotavirus RNA sequences. Our samp le-procesing technique and large-volume PCR protocol (reaction volume, 300 mu l) resulted in sufficient removal or dilution of inhibitors so that more than 95% of the samples could be assayed by PCR, Because of its sensitivit y for detecting viral nucleic acid sequences, PCR analysis should produce m ore positive results than cell culture analysis. Since either cell culture analysis or PCR can reveal only a ''snapshot" of the quality of the groundw ater being sampled, PCR seems to be a desirable rapid initial screening too l.