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Appl. Environ. Microbiol., Feb 1995, 531-537, Vol 61, No. 2
Copyright © 1995, American Society for Microbiology

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

KJ Schwab, R De Leon and MD Sobsey
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill 27599-7400, USA.

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.

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