Methodology

Nucleic-acid-based detection systems. The use of the polymerase chain reaction (PCR) as a diagnostic tool in veterinary diagnostic laboratories is now commonplace throughout the EU and elsewhere. However, most PCR tests being used are "in house" assays that vary in target specificity, sensitivity and specificity. In addition, internal controls for these tests vary from laboratory to laboratory. Fluorimeter-based, real-time sequence detecting technologies are beginning to replace gel-based conventional PCR in a number of diagnostic and research laboratories. This new technology uses sequence specific probes linked to a fluorescence energy transfer reaction (FRET). Specific PCR product can be detected in real-time, enhancing speed, sensitivity and specificity (McGoldrick et al., 1998; Leutenegger et al., 1999, 2001; Moody et al., 2000; Poddar, 2000; Alexandersen et al., 2001; Hadfield et al., 2001; Hofmann-Lehmann et al., 2001; Oleksiewicz et al., 2001; Clarke, 2002). Multiplexing of FRET PCR is also possible by the use of different fluorophors (Elnifro et al., 2000; Mackay et al., 2002). A further step in the evolution of nucleic acid detection is the possibility of fluorimeter-based detection of nucleic acid in a sample without thermocycling. This technology is novel and based on recently developed methods, like the Cleavase/Invader assay or the NASBA technology.

Efforts are also ongoing to develop simple PCR equipment that can be applied under field conditions or in simply equipped laboratories or abattoirs. The improvement of nucleic acid diagnostic methods is also ongoing with the development of novel methods for improved sample enrichment, DNA-tags, improved nucleic acid hybridisation methods, in situ PCR, macro- and micro array technologies (Anthony et al., 2001; Petrik, 2001; Ellis & Zambon, 2002; Ivnitski et al., 2003; Neill & Ridpath, 2003; Sugiyama et al., 2003; Zhao et al, 2003; Clewley, 2004).

Antigen-antibody detection systems are also undergoing modernisation and improvement. ELISA systems are being simplified the sensitivity is being increased and simplified dip-stick methodologies and other on-site technologies are being developed. In addition chemiluminescent and fluorometric immunoassays, chromatographic strips, colorimetric protein microarrays, antigen microarrays, biosensors are all being developed and evaluated for improved diagnostic procedures (Petrik, 2001; Mezzasoma et al., 2002; Neuman de Vegvar, 2003; Rider et al., 2003; Bacarese-Hamilton et al, 2004).