title: Modified concentration method for the detection of enteric viruses on fruits and vegetables by reverse transcriptase-polymerase chain reaction or cell culture.
abstract: Fruits and vegetables may act as a vehicle of human enteric virus if they are irrigated with sewage-contaminated water or prepared by infected food handlers. An elution-concentration method was modified to efficiently detect, by reverse transcriptase-polymerase chain reaction (RT-PCR) or by cell culture, contamination by poliovirus, hepatitis A virus (HAV), and Norwalk-like virus (NLV) of various fresh and frozen berries and fresh vegetables. The protocol included washing the fruit or vegetable surface with 100 mM Tris-HCl, 50 mM glycine, and 3% beef extract, pH 9.5 buffer, which favors viral elution from acid-releasing berries, supplemented with 50 mM MgCl2 to reduce the decrease in viral infectivity during the process. The viral concentration method was based on polyethylene glycol precipitation. Copurified RT-PCR inhibitors and cytotoxic compounds were removed from viral concentrates by chloroform-butanol extraction. Viruses from 100 g of vegetal products could be recovered in volumes of 3 to 5 ml. Viral RNAs were isolated by a spin column method before molecular detection or concentrates were filtered (0.22-microm porosity) and inoculated on cell culture for infectious virus detection. About 15% of infectious poliovirus and 20% of infectious HAV were recovered from frozen raspberry surfaces. The percentage of viral RNA recovery was estimated by RT-PCR to be about 13% for NLV, 17% for HAV, and 45 to 100% for poliovirus. By this method, poliovirus and HAV RNA were detected on products inoculated with a titer of about 5 x 10(1) 50% tissue culture infectious dose per 100 g. NLV RNA was detected at an initial inoculum of 1.2 x 10(3) RT-PCR amplifiable units. This method would be useful for the viral analysis of fruits or vegetables during an epidemiological investigation of foodborne diseases.
authors.affiliation: Unité de Virologie, Agence Française de Sécurité Sanitaire des Aliments, 22 rue Pierre Curie, D.P. 67, 94703 Maisons-Alfort cedex, France. e.dubois@afssa.fr
authors.forename: Eric
authors.initials: E
authors.lastname: Dubois
authors.forename: Cécilia
authors.initials: C
authors.lastname: Agier
authors.forename: Ousmane
authors.initials: O
authors.lastname: Traoré
authors.forename: Catherine
authors.initials: C
authors.lastname: Hennechart
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Merle
authors.forename: Catherine
authors.initials: C
authors.lastname: Crucière
authors.forename: Henri
authors.initials: H
authors.lastname: Laveran
mesh_terms: D002478:Cells, Cultured; D005506:Food Contamination; D005516:Food Microbiology; D005638:Fruit / Q000821:virology*; D017957:Hepatitis A Virus, Human / Q000302:isolation & purification*; D029322:Norovirus / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D017955:Poliovirus / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D012367:RNA, Viral / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014675:Vegetables / Q000821:virology*
chemical_list: D015345:Oligonucleotide Probes; D012367:RNA, Viral; D011092:Polyethylene Glycols