Optimization of Methods for Detecting Hepatitis A Virus in Food
- 131 Downloads
Hepatitis A virus (HAV) is currently recognized as an important human food borne pathogen, and it is one of the most resistant enteric RNA viruses, is highly infectious, and may lead to widespread outbreaks. The aim of this study was to optimize the methods to detect HAV from artificially contaminated food. To this end, strawberry and lettuce were experimentally contaminated with HAV suspension containing 6 × 106 copies/ml. After contamination, HAV persistence and washing procedure were evaluated at 0, 1, 3, 7, and 9 days of storage. Five elution buffers (PBS (pH 7.4)/0.1% Tween80; 50 mM glycine/3% (wt/vol) beef extract (pH 9.5); PBS (pH 7, 4); 25 mM glycine/0.1 Tween80; and 1 M sodium bicarbonate) were used to elute the virus, and qualitative and quantitative PCR were used for HAV detection. HAV was detected by qualitative and quantitative PCR using any of the five elution buffers, but PBS was the most effective. Even after washing, HAV was detected up to 9 days after contamination by quantitative PCR. Quantitative PCR was more sensitive than qualitative PCR since samples containing viral load lower than 1.4 × 103 copies/ml could not be detected by qualitative PCR. Quantitative PCR can be used for rapid detection of food borne viruses and will help in the monitoring and control of food borne disease.
KeywordsHepatitis A virus Food PCR Real-time PCR
The authors thank to Simone Teves and Renata Aloise for technical assistance. This work was supported by the Fundação Oswaldo Cruz (FIOCRUZ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
- Baptista, M. L., Silva, M., Lima, M. A., Yoshida, C. F., Gaspar, A. M., & Galler, R. (2006). Genetic variability of hepatitis A virus strain HAF-203 isolated in Brazil and expression of the VP1 gene in Escherichia coli. Memórias do Instituto Oswaldo Cruz, 101(7), 759–766.CrossRefPubMedGoogle Scholar
- Dubois, E., Hennechart, C., Deboosère, N., Merle, G., Legeay, O., Burger, C., et al. (2006). 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. International Journal of Food Microbiology, 25(108), 164–171.CrossRefGoogle Scholar
- Fiore, A. E. (2004). Hepatitis A transmitted by food. Food Safety CID, 38, 704–715.Google Scholar
- Guevremont, E., Brassard, J., Houde, A., Simard, C., & Trottier, Y.-L. (2006). Development of an extraction and concentration procedure and comparison of RT-PCR primer systems for the detection of hepatitis A virus and Norovirus GII in green onions. Journal of Virological Methods, 134(1–2), 130–135.CrossRefPubMedGoogle Scholar
- Hollinger, F. B., & Emerson, S. U. (2001). Hepatitis A virus. In B. N. Fields, D. N. Knipe, & P. M. Howley (Eds.), Virology (pp. 799–840). Philadelphia, PA: Lippincott Williams and Wilkins.Google Scholar
- Koopmans, M., von Bonsdorff, C. H., Vinje, J., de Médici, D., & Monroe, S. (2002). Foodborne viruses. FEMS Microbiol Reviews, 26(2), 187–205.Google Scholar
- Lukasik, J., Bradley, M. L., Scott, T. M., Hsu, W. Y., Farrah, S. R., & Tamplin, M. L. (2001). Elution, detection, and quantification of polio I, bacteriophages, Salmonella montevideo, and Escherichia coli O157:H7 from seeded strawberries and tomatoes. Journal of Food Protection, 64(3), 292–297.PubMedGoogle Scholar
- Morais, L. M., de Paula, V. S., Arantes, M. R., Oliveira, M. A., & Gaspar, A. M. C. (2006). Early infection and asymptomatic spread of hepatitis A virus in a public child care in Rio de Janeiro, Brazil: Should attending children under two years of age be vaccinated? Memórias do Instituto Oswaldo Cruz, 101(4), 401–405.CrossRefPubMedGoogle Scholar
- Sánchez, G., Bosch, A., & Pintó, R. M. (2007). Hepatitis A virus detection in food: Current and future prospects. Letters in Applied Microbiology, 45(1), 1–5. Review.Google Scholar
- Schwab, K. J., Neill, F. H., Fankhauser, R. L., Daniels, N. A., Monroe, S. S., Bergmire-Sweat, D. A., et al. (2000). Development of methods to detect ‘‘Norwalk-like viruses’’ (NLVs) and hepatitis A virus in delicatessen foods: Application to a food-borne NLV outbreak. Applied and Environmental Microbiology, 66(1), 213–218.CrossRefPubMedGoogle Scholar