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

• Published in: International journal of food microbiology Vol. 108; no. 2; pp. 164 - 171
• Main Authors: Dubois, Eric, Hennechart, Catherine, Deboosère, Nathalie, Merle, Ghislaine, Legeay, Odile, Burger, Christian, Le Calvé, Marie, Lombard, Bertrand, Ferré, Virginie, Traoré, Ousmane
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 25.04.2006; Elsevier
• Subjects: bacteriophages; Biological and medical sciences; chicory; Clinical Laboratory Techniques - standards; Coliphages - isolation & purification; Colony Count, Microbial - methods; Colony Count, Microbial - standards; cooperative research; endive; Enteric viruses; Enterovirus; Enterovirus - isolation & purification; Enumeration; Eruca sativa; Filtration; food contamination; Food Contamination - analysis; Food industries; Food Microbiology; food pathogens; Fruit and vegetable industries; Fundamental and applied biological sciences. Psychology; green leafy vegetables; Hepatitis A virus; Hepatitis A virus - isolation & purification; laboratories; Lactuca sativa; lettuce; Life Sciences; Linear Models; microbial detection; on-farm food safety; plate count; Poliovirus; Poliovirus - isolation & purification; ready-to-eat foods; Reproducibility of Results; RNA, Viral - analysis; Salad vegetables; salads; Sensitivity and Specificity; Valerianella locusta; Validation; Vegetables - virology; Viral Plaque Assay; virion; watercress; France; Validation; Filtration; Enumeration; Salad vegetables; Enteric viruses; Vegetables; Hepatitis A virus; Microbiological testing; RNA; Picornaviridae; Coliphage; Enterovirus; Plant leaf; Concentration; Laboratory; Method; Hepatovirus; Enumeration(counting); Virus; Salad; Phage
• DOI: 10.1016/j.ijfoodmicro.2005.11.007
• ISSN: 0168-1605

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.