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Development standardisation and harmonisation of novel multiplex nucleic acid tests for the detection of economically important viruses of farm animals


Conventional detection assays for viruses show several drawbacks like long diagnosis time or low sensitivity. The polymerise chain reaction (PCR) is powerful tool of rapid diagnosis offering new possibilities. Conventional PCR is still laborious and has disadvantages like contamination and lacks standardisation. Our aims are to develop novel, reliable, high-throughput multiplex PCR techniques for the improved detection of List A and other important viruses of farm animals. Multiplex enrichment and nucleic acid extraction procedures as well as internal controls will be applied in gel-based multiplex and fluorescence-based methods. These will provide speed and allow the economical investigation of high numbers of samples with minimal risk of contamination. The new procedures will be standardised and introduced as harmonised diagnostic methods for the rapid detection of economically important viruses of farm animals
A large variation of novel molecular methods has been developed for the improved diagnosis of economically important virus diseases of farm animals. The new methods include gel-based multiplex PCR assays, novel real-time PCR approaches, effective nucleic acid enrichment procedures and detection of viral nucleic acids without thermocycling. To assure the reliability of the new methods, internal controls were established. The gel-based multiplex PCR assays improved the simultaneous detection of several viruses in the same reaction vessel. The real-time PCR development included several methods, i.e. molecular beacons, TaqMan or Primer-Probe Energy Transfer (PriProET) techniques. Among molecular beacons SVDV detection system was the most suitable and it passed all preliminary tests. Both SVDV and VSV molecular beacon probes were optimised. Molecular beacons against CSFV, PRRSV, ADV and PPV (respiratory/reproductive cluster) as well as primers were designed. The cluster was divided into two assays due to limitations in detection capabilities. The first assay was designed to detect CSFV and ASFV, which correspond to the haemorrhagic cluster. The second assay detects PRRSV (European and US strains separately), ADV and PPV. Optimisation of the molecular beacon probes was finalised. The development of a real-time assay for FMDV, based on the newly invented PriProET system, was completed.

The ability of the assay to detect FMDV during clinical outbreak was assessed in clinical samples collected in an FMD endemic area in Uganda. The testing of clinical samples from Uganda showed that this new method is useful for diagnostic purposes. PriProET primers and probes against VSV and SVDV were optimised and tested. Individual assays for all three vesicular viruses were combined in a multiplex assay. Multiplexing of FMDV, SVDV and VSV were carried out. A gel-based multiplex-PCR method for the detection of viruses associated with hemorrhagic (CSFV/ASFV) and vesicular (SVDV/VSV/FMDV) List A diseases has been developed and standardized for its use in diagnostic laboratories as a routine assay. The work on immunocapture using capture probes has been completed. An ASFV Invader Squared Assay was established. An in house Invader assay was also designed using ordinary primer design software. Extraction controls have been developed. The project provided excellent conditions of technology transfer. Exchange of researchers was provided in the frame of the project, in order to transfer new information and technology, such as pre-cleaning of virus by capture probes on magnetic beads. Many of the new methods were introduced by partner laboratories (ring tests). The technology transfer also concerned the various equipments. For example, the PriProET system was originally developed for ABI PRISM 7700 equipment in Denmark.

Since the method was found very powerful in diagnostics, the partners adapted this technique to RotorGene2000, StratageneMX4000 and iCycler real-time PCVR machines in Sweden, Denmark and Hungary. Furthermore, the location of the donor dye in the primer was modified in order to operate by FRET system on iCycler. These results are very promising for further developments of the real-time based PCR assays using RotorGene 2000, Stratagene Mx4000 and iCycler. The technology transfer contributed to the introduction of the new diagnostic methods in various countries of the EU, including a new member Hungary. The development and international validation of the powerful diagnostic assays will contribute to an improved protection of the EU region from the introduction of eight important transboundary animal diseases.

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Ulls vaeg 2b
751 89 Uppsala

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Participants (5)