IMMUNOAFFINITY CONCENTRATION AND PURIFICATION OF WATERBORNE ENTERIC VIRUSES FOR DETECTION BY REVERSE-TRANSCRIPTASE PCR

To assess the risks from viral contamination of drinking-water supplie s, there is a clear need for methods to directly detect viral pathogen s, In this study, we developed a broad-spectrum immunocapture method f or concentration and purification of enteric viruses. The method invol ved indirect antibody capture (AbCap) of intact viruses followed by re lease of virion genomic RNA and reverse transcriptase PCR for amplific ation and oligoprobe hybridization for detection. The procedure involv ed concentrating enteric viruses from large volumes of water by standa rd filtration-elution techniques with 1MDS filters and 1 liter of 1% b eef extract-0.05 M glycine (BE/G) as an eluate. The BE/G eluate was co ncentrated and purified by polyethylene glycol (PEG) precipitation, Pr oCipitate (a commercially available protein precipitating reagent) pre cipitation, and a second PEG precipitation to a volume of approximatel y 500 mu l. Aliquots of the second PEG precipitate were further proces sed by RNA extraction, AbCap, or cell culture analysis for infectious viruses, The AbCap method was applied to 11 field samples of fecally c ontaminated surface water, Of the 11 samples, 9 were positive for ente ric viruses by the AbCap method; 4 of 11 samples were positive for ent eric viruses by direct RNA extraction of a small aliquot of the second PEG concentrate; and 4 of 11 samples were positive for enteric viruse s by measurement of cell culture infectivity. The results for enteric viruses were compared with those for standard bacterial and coliphage indicators of fecal contamination.