This article reports the development of a method to purify and concentrate intact virions from oyster extracts to a 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 101 to 105 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 mul.
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. (...)
Mots-clés Pascal : Mollusque et crustacé, Huître, Bivalvia, Mollusca, Invertebrata, Contamination biologique, Poliovirus 1, Enterovirus, Picornaviridae, Virus, Virus hépatite A, Virus Norwalk, Caliciviridae, Méthode, Hybridation moléculaire, Purification, Détection, Evaluation performance, Transcription inverse, Réaction chaîne polymérase
Mots-clés Pascal anglais : Shellfish, Oyster, Bivalvia, Mollusca, Invertebrata, Biological contamination, Poliovirus 1, Enterovirus, Picornaviridae, Virus, Hepatitis A virus, Norwalk virus, Caliciviridae, Method, Molecular hybridization, Purification, Detection, Performance evaluation, Reverse transcription, Polymerase chain reaction
Notice produite par :
Inist-CNRS - Institut de l'Information Scientifique et Technique
Cote : 96-0344698
Code Inist : 002A35B06. Création : 10/04/1997.