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Modified Concentration Method for the Detection of Enteric Viruses on Fruits and Vegetables by Reverse Transcriptase-Polymerase Chain Reaction or Cell Culture

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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-μm 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 × 101 50% tissue culture infectious dose per 100 g. NLV RNA was detected at an initial inoculum of 1.2 × 103 RT-PCR amplifiable units. This method would be useful for the viral analysis of fruits or vegetables during an epidemiological investigation of foodborne diseases.

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Document Type: Research Article

Affiliations: 1: Unite´ de Virologie, Agence Française de Sécurité Sanitaire des Aliments, 22 rue Pierre Curie, B.P. 67, 94703 Maisons-Alfort Cedex, France 2: Unite´ de Virologie, Agence Française de Sécurité Sanitaire des Aliments, 22 rue Pierre Curie, B.P. 67, 94703 Maisons-Alfort Cedex, France 3: Service d'Hygie`ne Hospitalière, Faculté de Médecine et de Pharmacie, 28 place Henri Dunant, 63000 Clermont-Ferrand, France 4: Unite´ de Virologie, Agence Française de Sécurité Sanitaire des Aliments, 22 rue Pierre Curie, B.P. 67, 94703 Maisons-Alfort Cedex, France

Publication date: 2002-12-01

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