Kj. Schwab et al., IMMUNOAFFINITY CONCENTRATION AND PURIFICATION OF WATERBORNE ENTERIC VIRUSES FOR DETECTION BY REVERSE-TRANSCRIPTASE PCR, Applied and environmental microbiology, 62(6), 1996, pp. 2086-2094
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.