• Respiratory (CSFV, ASFV, PRRSV, ADV)
• Reproductive (CSFV, ASFV, PRRSV, ADV, PPV)
• List A, haemorrhagic (CSFV, ASFV)
• List A, vesicular (SVDV, FMDV, VSV)

Summary of the main objectives of the Project

Objective 1: The development, standardisation and harmonisation of "conventional" gel-based multiplex PCR tests for detection of virus infections in farm animals.

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 developed and standardised for its use in diagnostic laboratories as a routine assay. The other five target viruses associated with reproductive disorders (ASF, CSF, ADV, PPV and PRRSV) were divided into two groups: ASF/CSF/ADV and PRRSV/PPV/ADV according to conventional, gel-based multiplex PCR assays. The assay was optimised and evaluated. Another gel-based PCR assay that can detect and differentiate the American and European subtypes of PRRSV was developed and optimised as well. In summary, individual and multiplex gel-based PCR assays that cover all eight swine viruses that belong to the List A (OIE classification) were established and can be used in diagnostic laboratories by trained personal as routine diagnostic methods.

Objective 2: The development, standardisation and harmonisation of fluorimeter-based real time (non-gel based) multiplex PCR tests for detection of virus infections in farm animals.

The conventional PCR is currently being replaced by fluorimeter-based, real-time sequence detecting technology in a few laboratories throughout the EU. This new technology dispenses with the need for gel electrophoresis to identify the PCR product and uses sequence-specific probes linked to fluorescence resonance energy transfer (FRET) reactions. Specific product can be detected in real time, enhancing speed, sensitivity and specificity. Multiplexing with FRET and PCR is now possible with the production of new fluorophores and multi-channel instrumentation.

Advanced fluorimeter-based single and multiplex detection of economically important viruses have been developed and optimised. Primer-probe energy transfer (PriProET) system and molecular beacon assays were used. PriProET is a flexible alternative system, which in some cases is 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. At present, PriProET assays for the three vesicular 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 and specificity of the multiplex assays were closely correlated to that of the individual PriProET assays. Molecular beacon probes have been developed and optimised for the vesicular, reproductive, respiratory and hemorrhagic clusters (SVDV, VSV, ASFV (work ongoing), CSFV, PRRSV, ADV and PPV).

Objective 3: The development of multiplex nucleic acid enrichment procedures to increase sensitivity of multiplex PCR.

Increased sensitivity of diagnostic testing, without loss of specificity, is always a legitimate target for research. This project addressed this target for the multiplex PCR tests proposed by developing antibody and nucleic acid capture technology. 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) and an RNA virus (CSFV). 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.

Objective 4: Development of methodology for the detection of viral nucleic acid without thermocycling (i.e., Invader technique) using a DNA model.
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 assay. The assay is a linear, isothermal (63¼C) signal amplification system, which targets the VP73 gene of ASFV. The method is based on the hybridisation of target specific invader and hybridisation probes to the target of interest followed by structure specific cleavage and signal generation by the Cleavase XI enzyme. The Invader assays were successfully evaluated on a real time PCR machine (iCycler, Biorad). The assay works well with different ASFV strains and also on clinical material. The possibility of designing an Invader assay “in house” was also explored without success.

Objective 5: Production and application of a library of internal controls for PCR technology to be applied to the above tests. These controls should allow EU wide standardisation and harmonisation of this technology.

The project developed a library of internal controls for the "conventional" gel-based multiplex PCR assay and fluorimeter-based, real-time sequence detecting technology linked to FRET reactions.