Methodology
This project developed and standardiseed such tests:
Conventional (gel-based) multiplex PCR assays are currently
being applied, primarily in experimental conditions, to the
detection of more than one infectious agent in a clinical
sample. Although these tests offer advantages of speed, sensitivity
and economy they still suffer from lack of harmonised controls
to apply across diagnostic laboratories. This project developed
specific packages of multiplex, gel-based PCR assays (Objective
1) and a library of internal controls for these assays (Objective
5).
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 reactions (FRET probes). 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.
This project was harness this evolving technology and developed fluorimeter-based
multiplex PCR tests for a number of viral infections of farm animals, based on
FRET probes (Objective 2). As above, relative internal controls for these tests
was developed and harmonised (Objective 5).
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 Clevase/Invader assay.
This project attempted to apply this novel technology to the development of assays
for ASFV (Objective 4).
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, i.e., anthraquinone-based photochemical method. The covalent binding
is very stable and it is therefore possible to mix beads coated with monoclonal
antibodies against several viruses with beads coated with nucleotides specific
for the same viruses in one tube. Pre-treating the clinical samples with this
tool allowed the simultaneous or sequential enrichment of virions and free viral
nucleic acids in one tube (Objective 3).
