Molecular diagnostics of viral animal diseases.: Summary article

This review article summarises the achievements and experiences obtained in a molecular diagnostic research and development laboratory during the last two decades. The laboratory started the diagnostic application of various polymerase chain reaction (PCR) methods as early as 1988. Approximately 35 nested PCR assays are used today for routine diagnosis of a wide range of diseases of domestic and wild animals. Specific precautions were elaborated to prevent false positive results, while the false negatives are avoided by using internal controls (mimics). Recently, the nested PCR assays are replaced in the routine diagnosis by various methods of real-time PCR, such as TaqMan, Molecular Beacon and the Primer-Probe Energy Transfer System. Multiplex PCR assays are developed to provide a complex and rapid identification of a range of disease syndrome complexes. For example, a multiplex PCR system has been developed for the diagnosis of vesicular diseases of swine. The,vesicular multiplex PCR" is able to detect simultaneously the viruses of foot-and-mouth disease, swine vesicular disease and vesicular stomatitis, within several hours. Diagnosis is further improved by the use of nucleic acid extraction and pipetting robots, coupled to the real-time PCR machines. By this way the molecular diagnostic procedures are automated, providing high diagnostic throughput, rapidity and reliability. The direct nucleotide sequence analysis of the PCR products has created possibilities to determine the genetic relationships of virus variants, to trace the routes of infections in studies of molecular epizootiology. To harmonise the molecular diagnosis word-wide, the five stages of OIE validation are followed.