Concentration and purification of beef extract mock eluates from water samples for the detection of enteroviruses, hepatitis A virus, and Norwalk virus by reverse transcription-PCR.
In this study we developed a concentration and purification procedure to facilitate reverse transcription (RT)-PCR detection of enteric viruses in water sample concentrates obtained by conventional filter adsorption-elution methods. One liter of beef extract-glycine eluate with or without humic acid and seeded with poliovirus type 1, hepatitis A virus, and Norwalk virus was used as a model system, and the eluent was further processed for RT-PCR compatibility. The sample concentration and purification procedures which we used included polyethylene glycol precipitation, Pro-Cipitate precipitation, a second polyethylene glycol precipitation, spin column chromatography, and ultrafiltration. The sample volumes were reduced from 1 liter to 20 to 50 microliters, and the samples were purified enough so that viruses could be detected by the RT-PCR. The ability to detect low levels of enteric viruses by molecular techniques was compared directly with the ability to detect enteric viruses by cell culture infectivity procedures. As little as 3 PFU of poliovirus type 1 in an initial 1 liter of mock eluate was detected by the RT-PCR.
Schwab
K J
KJ
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill 27599-7400, USA.
De Leon
R
R
Sobsey
M D
MD
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D011232:Chemical Precipitation; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D006507:Hepatovirus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D009663:Norwalk virus / Q000235:genetics / Q000302:isolation & purification*; D015345:Oligonucleotide Probes; D011092:Polyethylene Glycols; D016133:Polymerase Chain Reaction / Q000379:methods*; D014773:Virology / Q000379:methods; D014871:Water Microbiology
D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D011092:Polyethylene Glycols