Conclusions

The main aim of the project was to develop, harmonise and standardise multiplex PCR technology (gel-based, fluorimeter-based and Invader-based) for the detection of eight animal viruses of economic importance. The studied viruses were African swine fever virus (ASFV), classical swine fever virus (CSFV), Aujeszky's disease virus (ADV), foot and mouth disease virus (FMDV), vesicular stomatitis virus (VSV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV) and swine vesicular disease virus (SVDV). The viruses were grouped into four clusters, based on possible clinical presentation: respiratory (CSFV, ASFV, PRRSV, ADV), reproductive (ASFV, CSFV, ADV, PPV, PRRSV), haemorrhagic (ASFV, CSFV) and a vesicular (SVDV, FMDV, VSV).

In order to improve diagnostic developments, a database of viral sequences was established. The aim was to collect all possible sequences of the given viruses, even those parts of the viral genome, which were not readily considered to be optimal targets for diagnostic PCR. The database was established in Genedoc format and consists of downloaded sequences with GenBank accession numbers and references. Conclusion: the combined database, containing sequence data from eight different viruses is a large, uniform and valuable tool for the work in the other work packages.

Optimisation and evaluation of gel-based PCR assays for the individual and multiplex detection of viruses associated with haemorrhagic (CSFV/ASFV) and vesicular (SVDV/VSV/FMDV) List A diseases was completed. The assays were standardised for use in diagnostic laboratories as routine diagnostic methods. The other five target viruses associated with reproductive disorders (ASF, CSF, ADV, PPV and PRRSV) were divided onto two groups: ASF/CSF/ADV and PRRSV/PPV/ADV according to conventional, gel-based multiplex PCR assays. These assays were optimised and evaluated as well. Conclusion: the individual and multiplex gel-based PCR assays detect all the eight swine viruses that belong to the List A (OIE classification) can be used in diagnostic laboratories by trained personal as routine diagnostic methods.

Advanced fluorimeter-based single and multiplex detection assays of economically important viruses have been developed and optimised. Molecular beacon probes have been developed and optimised for all four clusters of viruses (see above). A new fluorimeter-based real-time PCR assay, based on primer-probe energy transfer (PriProET) principle, was developed by Partner 3 and adapted by other Partners. PriProET is a flexible alternative system, which is in some cases superior to TaqMan or molecular beacons in real-time PCR. Development of PriProET assays for each of the vesicular (FMDV, VSV, SVDV) and haemorrhagic (CSFV, ASFV) viruses is completed. PriProET assays for the viruses in the vesicular cluster were combined in a single multiplex assay and multiplexing of FMDV, SVDV, VSV as well as CSFV, ASFV were carried out. The specificity of the multiplex assay for each virus was the same as for the individual PriProET assays. The sensitivity of the multiplex PCR for vesicular and haemorrhagic clusters of the viruses was similar to that of the individual PriProET PCR assays. Conclusion: the fluorimeter-based real-time PCR assays, developed in this project, provide powerful novel tools for the improved diagnosis of the eight targeted diseases of swine.

The increased sensitivity of diagnostic assays, without loss of specificity, is always a legitimate target for R&D laboratories. This project addressed this target for the multiplex PCR tests proposed by developing antibody and nucleic acid capture technologies. Novel routes and combinations of coupling the capturing molecules were applied. The work on the pre-cleaning of samples prior to PCR using magnetic beads was performed. Streptavidine-coated beads tied to biotin-labelled nucleic acid probes were analysed to catch a DNA virus (ASFV as a model) and an RNA virus (CSFV as a model). Several different approaches were analysed and a protocol for extraction of RNA or DNA was developed. Using nucleic acid analogues for catch probes were in a few cases superior to DNA, but generally the constitution of the nucleic acid catch probe did not change the catching efficacy. Conclusion: the novel enrichment methods provide further support to the improved diagnosis of the eight targeted diseases in swine.

A further step in the evolution of nucleic acid detection is the possibility of fluorimeter-based visualisation of nucleic acid, without thermocycling. This technology is novel and based on the recently developed Cleavase/Invader principle. The assay is a linear, isothermal (63¼C) signal amplification system, which targets the VP73 gene of ASFV (used as a model). It is based on the hybridisation of target specific invader and hybridisation probes to the target of interest and subsequent signal generation via the action of the structure specific flap endonuclease Cleavase XI enzyme. The Invader assays were successfully evaluated on a real-time PCR machine (iCycler, BioRad). Conclusion: the isothermal amplification assay works well with different ASFV strains and also on clinical material.

A library of internal controls for the "conventional" gel-based multiplex PCR assays and fluorimeter-based real-time sequence detecting technology linked to fluorescence resonance energy transfer (FRET) reactions were developed during a lifetime of the project.

In order to standardise diagnostic assays and harmonise their use internationally, the new gel-based and the real-time PCR assays were evaluated by following the OIE rules of validation. The assays were validated by performing ring tests. Conclusion: the project significantly contributed to the international standardisation of the molecular diagnostic assays.

In summary, the project has reached the goals: novel multiplex nucleic acid tests were developed for the improved diagnosis of eight economically important diseases of farm animals. The project brought together a powerful Consortium of six European laboratories, including research centres, universities and one SME. The developed new diagnostic assays have been introduced in the routine diagnostic services of the partner countries and the results were disseminated worldwide. The project contributed to the strengthening of European research and established a large network of collaboration.