Experiences of an OIE Collaborating Centre in molecular diagnosis of transboundary animal diseases: a review.

The highly contagious transboundary animal diseases (TADs), e.g., foot-and-mouth disease (FMD), classical swine fever (CSF), African swine fever (ASF) and highly pathogenic avian influenza (HPAI) are regularly occurring and re-occurring on various continents, causing severe losses. This epidemiological situation indicates the urgent need for the development of powerful, robust and high capacity new diagnostic methods in order to detect and identify the causative agents very rapidly. This report is on the experiences of an OIE Collaborating Centre and those of the MULTIPLEX-PCR and the LAB-ON-SITE EU project consortia with the deveopment of novel methods for the improved molecular diagnosis of a range of viral diseases. Thermal amplification based real-time PCR methods (e.g.,TaqMan, Molecular Beacons, Primer-Probe Energy Transfer, and Light Upon eXtension (LUX) fluorogenic primers), and amplification without thermocycling have been elaborated for the improved diagnosis of TADs, such as FMD, swine vesicular disease, vesicular stomatitis, CSF, ASF, HPAI and Newcastle disease (ND). The simultaneous detection of various pathogens in a disease complex is facilitated by the development of multiplex PCR packages. By introducing nucleic acid extraction and pipetting robotics, together with the multi-channel real-time PCR machines, the molecular diagnostic procedures have become rapid, robust and automated. Quality control is strengthened by special precautions to avoid false positive and false negative results. By following the steps of OIE standardisation and validation, the diagnostic PCR assays have become nationally and nternationally standardised and harmonised. The development of additional methods, like padlock probes and microarrays, is further improving the arsenal of nucleic acid based novel molec ular diagnostic tests for TADs.