Virion concentration method for the detection of human enteric viruses in extracts of hard-shelled clams.
A method to extract and concentrate intact human enteric viruses from oyster extracts for detection using reverse transcription-polymerase chain reaction (RT-PCR) was applied to hard-shelled clams (mercenaria mercenaria). Fifty-gram clam samples were processed by an adsorption-elution-precipitation method and then seeded with 10(1) to 10(5) PFU to poliovirus 1 (PV1) and/or hepatitis A virus (HAV). Seeded viruses in extracts were purified by fluorocarbon (Freon) extraction and concentrated by polyethylene glycol (PEG) precipitation and elution. Efficiency of virion recovery from PEG precipitates was dependent upon PEG concentration and elution buffer volume, with optimized variable yielding recoveries as high as 99% for PV1 and 45% for HAV, as evaluated by cell culture infectivity assay. To further concentrate viruses, remove inhibitors, and reduce sample volumes, the protein-precipitating agent Pro-Cipitate was used in an adsorption-elution-precipitation scheme. The final concentrate was of low volume (< 1 ml) and directly compatible with viral genomic amplification using RT-PCR. When extracts from 50-g clam samples were seeded and processed by the combined concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 10(3) PFU for PV1 and HAV. Corresponding virus recoveries based on cell culture infectivity were 7 to 50% and 0.3 to 8% for pV1 and HAV, respectively, when extracts of clams were artificially contaminated with the Norwalk virus, direct detection of virion RNA using RT-PCR and subsequent oligoprobe hybridization was possible at levels as low as 450 RT-PCR amplifiable units of the Norwalk virus per extract of 50-g clam sample.
Food Science Department, North Carolina State University, Raleigh 27695-7624, USA.
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D000818:Animals; D049872:Bivalvia / Q000821:virology*; D005617:Chlorofluorocarbons, Methane; D017957:Hepatitis A Virus, Human / Q000302:isolation & purification*; D009663:Norwalk virus / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D017955:Poliovirus / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D016133:Polymerase Chain Reaction / Q000379:methods; D012758:Shellfish / Q000821:virology*; D014773:Virology / Q000379:methods*
D005617:Chlorofluorocarbons, Methane; D015345:Oligonucleotide Probes; D011092:Polyethylene Glycols