B04001: The development of improved simplified and standardised PCR based techniques for the detection of Norovirus and hepatitis A virus in molluscan shellfish
Friday 30 April 2004
This research project aims to adapt existing methods to establish a simple, reliable, robust and well standardised method for Norovirus detection.
Study Duration: May 2000 to April 2003
Contractor: Centre for Environment, Fisheries and Aquaculture Sciences (CEFAS)
Background
Filter-feeding bivalve molluscan shellfish (oysters, mussels, etc) concentrate microbial contaminants occurring in their growing waters and may present a health hazard when consumed raw or lightly cooked. Human enteric viruses, occurring as a consequence of sewage contamination of growing waters, are the pathogens most frequently associated with illness following shellfish consumption. Control measures for such contamination are hindered by the current absence of routine test methods for detection of such viruses in shellfish.
The successful detection of enteric viral pathogens such as Norovirus (NV) and hepatitis A in molluscan shellfish relies on Polymerase Chain Reaction (PCR) based techniques. The presence of NVs can be readily demonstrated in shellfish from polluted harvesting areas, but detection in less polluted shellfish products is more difficult due to lower levels of virus being present.
Research Approach
The overall objective of this project is to develop and establish PCR methods for the detection of human enteric viral pathogens (Norovirus and hepatitis A virus) contaminating molluscan shellfish that are as simple as possible and that are reliable, robust and well standardised. Achievement of this objective would enable a wider uptake of such test methods by more routine food control laboratories.
Results and findings
This project has successfully standardised and optimised a method for detection of Norovirus (NV) and hepatitis A (HAV) in bivalve molluscan shellfish. The method was applied to a large evaluation panel of 242 shellfish over an 18 month period. Approximately 50% of samples were positive for NV, however, this cannot be regarded as representative of general levels of NV contamination in harvesting areas as this evaluation panel was biased towards viral positive samples. NV was confirmed by sequencing in 73 samples, approximately 60% of those selected for confirmation, with a variety of NV sequences seen. Virtually all NV strains were successfully identified in relation to a clinical counterpart and no obvious veterinary strains were identified. The HAV assay performed successfully in European ring trials.
A real-time, TaqMan based, assay was developed and evaluated and shows considerable potential for replacement of the conventional nested Polymerase Chain Reaction (PCR) format when pan-specific TaqMan primers and probes for NV become available. TaqMan chemistry is well standardised and has significant advantages of speed, robustness and labour costs. The TaqMan assay performed well in comparison with conventional nested PCR showing equivalent or improved sensitivity for detection of NV in shellfish. Real-time PCR, TaqMan, also offers the potential for NV quantitation, which could be critical for interpretation of field data in the future.
Commercial kits were evaluated to simplify and standardise nucleic acid extraction/purification stages of the assay. However, none were found to perform as well as the in-house assay. A kit supplied by Biorad inc. gave the best performance and, with further small modifications, may prove suitable. The heteroduplex mobility assay was investigated as an alternative to sequencing for NV confirmation. However, assay interpretation was confounded by the multiple NV strains commonly found in contaminated shellfish. A commercial NV Enzyme Linked Immunosorbent Assay (ELISA) was evaluated for application to shellfish, however, it was found to be too insensitive to replace PCR based methods.
Dissemination information
Project completed - Final report is awaited.
Formiga-Cruz M., Lees D.N., Henshilwood K., Allard A., Hernoth B., Vantarakis A., Papapetropoulou M. and Girones R. (2002). Distribution of viral pollution of shellfish in Europe. Applied and Environmental Microbiology, 68 (12) 5990-5998.
Formiga-Cruz M., Allard A. K., Condin-Hansson A.-C., Henshilwood K., Hernoth B. E., Jofr J., Lees D. N., Lucena F., Papapetropoulou, Rangdale R. E., Tsibouxi A., Vantarakis A. and Girones R. (2003). Evaluation of potential viral contamination in shellfish with applicability to diverse geographical areas. Applied and Environmental Microbiology, 69 (3) 1337-1874.
D.N. Lees, K. Henshilwood, R. E. Rangdale and W. J. Dore (2003). Viruses and bivalve shellfish: the development of sanitary controls. In: Molluscan Shellfish Safety. A. Villalba, J.L. Romalde, B. Reura & R. Beiras (eds).
Ignacio Torrado, Kathleen Henshilwood, David Lees & Jesus L. Romalde (2003). Detection Of Enteric Viruses In Shellfish By The Nested-PCR Method, And Comparision With F-Specific Rna Bacteriophage And Escherichia Coli Counts. In: Molluscan Shellfish Safety. A. Villalba, J.L. Romalde, B. Reura & R. Beiras (eds).
Katheen Henshilwood, William Dore, Sarah Anderson and David Lees (2003). Investigation Of Norwalk Like Virus Elimination During Depuration Using A Real Time Quantitative PCR. In: Molluscan Shellfish Safety. A. Villalba, J.L. Romalde, B. Reura & R. Beiras (eds).
Henshilwood, K., Dore, W., Lees, D. (2001). The use of molecular techniques for monitoring enteric virus contamination of shellfish harvesting areas. Journal of Clinical Virology, 22:157.