A virion concentration method for detection of human enteric viruses in oysters by PCR and oligoprobe hybridization.
This article reports the development of a method to purify and concentrate intact virions from oyster extracts to volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase PCR (RT-PCR) and confirmation by oligonucleotide probe hybridization. Fifty-gram oyster samples were processed by an adsorption-elution -precipitation method and then seeded with 10(1) to 10(5) PFU of poliovirus type 1 (PV1) or hepatitis A virus (HAV). Seeded viruses in oyster extracts were purified by fluorocarbon extraction and concentrated by polyethylene glycol (PEG) precipitation and elution. Virus recovery after elution of PEG precipitates was dependent upon PEG concentration and averaged 60% for PV1 and 40% for HAV. The next processing step used the protein-precipitating agent Pro-Cipitate (Affinity Technology, Inc., Brunswick, N.J.) in an adsorption-elution -precipitation scheme to further concentrate viruses and reduce sample volumes to 100 microliter. Oyster extracts processed by Pro-Cipitate adsorption-elution-precipitation were directly compatible with RT-PCR and yielded virus recoveries of > 80% for both PV1 and HAV. When extracts from 50-g oyster 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 PFU for both PV1 and HAV and with low levels of Norwalk virus. Virus recoveries based on cell culture infectivity were 25 to 35% for PV1 and 5 to 10% for HAV. When tested on artificially contaminated raw oysters, the combined method successfully detected > or = 10(3) PFU of PV1 and HAV and 10(5) RT-PCR-amplifiable units of Norwalk virus. Virus detection by RT-PCR and cell culture infectivity was consistent and well correlated among replicate samples and at different virus titers. The procedure developed in this study is rapid, sensitive, and effective for the direct detection of enteric viruses in oysters by RT-PCR.
Jaykus
L A
LA
Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill 27599-7400, USA.
De Leon
R
R
Sobsey
M D
MD
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; 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; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics; D010125:Ostreidae / Q000821:virology*; D017955:Poliovirus / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000821:virology*; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes