title: Identification of Norwalk virus in artificially seeded shellfish and selected foods.
abstract: A rotavirus dsRNA purification protocol was adapted to extract Norwalk ssRNA from artificially contaminated shellfish, and a sensitive reverse transcription-polymerase chain reaction assay for Norwalk virus was devised to identify an estimated 20-200 genomic copies. The technique includes deproteinization with guanidinium isothiocyanate, adsorption of RNA to hydroxyapatite, and sequential precipitation with cetyltrimethylammonium bromide and ethanol. The protocol allows high recovery of viral RNA free of enzymatic inhibitors from oysters, clams, and a variety of food matrices. Norwalk virus sequences were copied and amplified by using primers selected from the polymerase gene. Digestion of the amplified products with restriction enzymes ensured the specificity of the test. This rapid and sensitive assay may significantly improve the prospect for the routine screening of the uncultivatable Norwalk virus in food stuffs.
authors.affiliation: Division of Molecular Biological Research and Evaluation, Food and Drug Administration, Washington, DC 20204.
authors.forename: V
authors.initials: V
authors.lastname: Gouvea
authors.forename: N
authors.initials: N
authors.lastname: Santos
authors.forename: M do C
authors.initials: Mdo C
authors.lastname: Timenetsky
authors.forename: M K
authors.initials: MK
authors.lastname: Estes
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005516:Food Microbiology; D005517:Foodborne Diseases / Q000209:etiology; D005759:Gastroenteritis / Q000209:etiology; D006801:Humans; D008969:Molecular Sequence Data; D009663:Norwalk virus / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D010710:Phosphates; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D017680:Potassium Compounds; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000821:virology*
chemical_list: D017931:DNA Primers; D010710:Phosphates; D017680:Potassium Compounds; D012367:RNA, Viral; C013216:potassium phosphate