Detection and quantification by real-time RT-PCR of hepatitis A virus from inoculated tap waters, salad vegetables, and soft fruits: characterization of the method performances.

Water, salad vegetables and fruits exposed to fecal contamination may cause outbreaks of hepatitis A. A protocol of viral concentration by filtration on electronegative membrane filter and a protocol based on a viral elution in Tris-glycine buffer, pH 9.5 with concentration by polyethylene glycol precipitation were associated with real-time, reverse transcriptase-PCR to detect hepatitis A virus (HAV) artificially inoculated in 2 l of tap water, or on 25 g of fruits or salad vegetables. These methods were characterized by an intra-laboratory study using the international standard ISO 16140 on five types of tap water, six types of fruit and five types of salad vegetable. Linear regression models describing the quantitative reactions were good fits to data, and the variances of results were constant in the whole range of viral concentrations tested, which was from about 1.7 to 5.7 log plaque-forming units (PFU) per 2 l of tap water, from about 2.0 to 4.5 log PFU/25 g of fruits, and from 1.5 to 3.5 log PFU/25 g of salad vegetables. Fractions of inoculated viruses recovered were estimated to be about 20% for tap water, about 16% for salad vegetables, and about 7% for fruits. The probability of detecting positive samples was 50% (the critical level of detection) when 2 l samples of tap water were inoculated with 0.7 log PFU of HAV, 25 g samples of iceberg lettuce were inoculated with 1.0 log PFU of HAV, and 25 g samples of fresh and frozen raspberries were inoculated with 1.0 and 1.5 log PFU of HAV, respectively.

Intra-laboratory validation of a concentration method adapted for the enumeration of infectious F-specific RNA coliphage, enterovirus, and hepatitis A virus from inoculated leaves of salad vegetables.

Salad vegetables exposed to fecal contamination may cause outbreaks of hepatitis or gastro-enteritis if they are eaten raw. A procedure, based on elution with phosphate-buffered saline and concentration by filtration through membrane filters, was developed for the recovery of enteric viruses from salad leaves. The method was evaluated using lettuce leaves inoculated with hepatitis A virus (HAV), poliovirus, and MS2 bacteriophage. In addition, this method was validated by an intra-laboratory study using leaves of various salad vegetables inoculated with MS2 phage. The French standard NF V 03-110 was used to establish the general principle and the technical protocol of the validation procedure. Linear regression models describing the quantitative reactions were good fits to data in the whole range of viral concentrations tested, which was from about 1 to 4 log plaque-forming units (PFU) per 25 g of lettuce. The fractions of inoculated viruses recovered were estimated to be about 64% for HAV, 18% for poliovirus, and 29% for MS2. No significant effect of the food matrix was found using various types of salad vegetable (butter lettuce, iceberg lettuce, romaine lettuce, witloof chicory, curly endive, corn salad, rocket and watercress). Moreover, the variance of the results was constant for all levels of virus contamination within the experimental range. Intermediate reproducibility experiments were also performed to allow calculation of the uncertainty factor, which was found to be 0.58 log PFU/25 g. When used in association with phage enumeration, this validated procedure is rapid enough to be used for screening salad vegetables for evaluation of the efficacy of processes for control of pathogenic microorganisms on such foods.

Modified concentration method for the detection of enteric viruses on fruits and vegetables by reverse transcriptase-polymerase chain reaction or cell culture.

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.