TY - JOUR
T1 - Immunoaffinity concentration and purification of waterborne enteric viruses for detection by reverse transcriptase PCR
AU - Schwab, Kellogg J.
AU - De Leon, Ricardo
AU - Sobsey, Mark D.
PY - 1996
Y1 - 1996
N2 - To assess the risks from viral contamination of drinking-water supplies, there is a clear need for methods to directly detect viral pathogens. In this study, we developed a broad-spectrum immunocapture method for concentration and purification of enteric viruses. The method involved indirect antibody capture (AbCap) of intact viruses followed by release of virion genomic RNA and reverse transcriptase PCR for amplification and oligoprobe hybridization for detection. The procedure involved concentrating enteric viruses from large volumes of water by standard filtration-elution techniques with 1MDS filters and 1 liter of 1% beef extract-0.05 M glycine (BE/G) as an eluate. The BE/G eluate was concentrated and purified by polyethylene glycol (PEG) precipitation, ProCipitate (a commercially available protein precipitating reagent) precipitation, and a second PEG precipitation to a volume of approximately 500 μl. Aliquots of the second PEG precipitate were further processed by RNA extraction, AbCap, or cell culture analysis for infectious viruses. The AbCap method was applied to 11 field samples of fecally contaminated surface water. Of the 11 samples, 9 were positive for enteric viruses by the AbCap method; 4 of 11 samples were positive for enteric viruses by direct RNA extraction of a small aliquot of the second PEG concentrate; and 4 of 11 samples were positive for enteric viruses by measurement of cell culture infectivity. The results for enteric viruses were compared with those for standard bacterial and coliphage indicators of fecal contamination.
AB - To assess the risks from viral contamination of drinking-water supplies, there is a clear need for methods to directly detect viral pathogens. In this study, we developed a broad-spectrum immunocapture method for concentration and purification of enteric viruses. The method involved indirect antibody capture (AbCap) of intact viruses followed by release of virion genomic RNA and reverse transcriptase PCR for amplification and oligoprobe hybridization for detection. The procedure involved concentrating enteric viruses from large volumes of water by standard filtration-elution techniques with 1MDS filters and 1 liter of 1% beef extract-0.05 M glycine (BE/G) as an eluate. The BE/G eluate was concentrated and purified by polyethylene glycol (PEG) precipitation, ProCipitate (a commercially available protein precipitating reagent) precipitation, and a second PEG precipitation to a volume of approximately 500 μl. Aliquots of the second PEG precipitate were further processed by RNA extraction, AbCap, or cell culture analysis for infectious viruses. The AbCap method was applied to 11 field samples of fecally contaminated surface water. Of the 11 samples, 9 were positive for enteric viruses by the AbCap method; 4 of 11 samples were positive for enteric viruses by direct RNA extraction of a small aliquot of the second PEG concentrate; and 4 of 11 samples were positive for enteric viruses by measurement of cell culture infectivity. The results for enteric viruses were compared with those for standard bacterial and coliphage indicators of fecal contamination.
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U2 - 10.1128/aem.62.6.2086-2094.1996
DO - 10.1128/aem.62.6.2086-2094.1996
M3 - Article
C2 - 8787407
AN - SCOPUS:0029888559
SN - 0099-2240
VL - 62
SP - 2086
EP - 2094
JO - Applied and environmental microbiology
JF - Applied and environmental microbiology
IS - 6
ER -