Block 1. Development and validation of real-time PCR for HAV and NV (0-18 months).

Robust, reliable, sensitive, specific, standardised and quantitative second generation real-time diagnostic RT-PCR assays for HAV (WP3.3.1) (partners 27.UB, 23.ISS) and NV (WP3.3.2) (partners 23.ISS, 65.RIVM, 38.UH-HI) will be developed for, primarily, the TaqMan instrumentation platform. Primer/probe design will be accomplished using commercial software and both sequence databanks and partner sequence information. Primer/probe sets will be evaluated against banks of virus reference strains and against clinical material. For Norovirus a currently funded DG Sanco project (finishing December 2003) will contribute real-time primer/probe sets. For both viruses final evaluation will be performed on shellfish panels. It is possible that broadly reactive primer/probe sets cannot be developed for Norovirus beacause of the diversity of viral strains. A contingency in this case will be to develop a family of primer/probe sets that could be used in a multiplex assay format.

Block 2. Development of control reagents and standards (0-18 months).

Assay reagents such as PCR internal controls and reference standards will be developed for HAV (WP3.1.1) (partners 27.UB, 23.ISS) and NV (WP3.1.2) (partners 23.ISS, 65.RIVM, 38.UH-HI,) using both classical and molecular techniques. This will include the evaluation of commercially available reagents such as ‘armoured RNA’.

Block 3. Interlaboratory evaluation and validation (19-36 months).

Standardised assay methods will be evaluated and validated among a number of EU laboratories. Standard panels of ‘spiked’ and naturally contaminated shellfish will be assayed blind to establish interlaboratory assay performance. Validation laboratories will specifically include EU National Reference laboratories and will follow ISO method validation protocol requirements. Validation evaluation will include naturally contaminated samples, seasonal and geographical factors, effects of differing shellfish pollution status, shellfish species, etc. As part of the validation exercise surveillance of virus in imported and EU produced shellfish, and before and after depuration, will provide data for risk assessment purposes (Partners 27.UB, 3.IFREMER, 23.ISS).

Block 4. Derivation of standard protocols, quality assurance to ISO 17025 standard, ISO method (37 –54 months).

Following validation standard protocols and quality assurance procedures will be developed and a standard method proposed to ISO. The project will include activities relating to ISO working groups and the establishment of a standard ISO method (Partners 27.UB, 3.IFREMER, 23.ISS, 38.UH-HI, 44.USC).