Development of a highly sensitive real-time RT-PCR protocol for the detection of Classical swine fever virus independent of the 5′ untranslated region

SummaryClassical swine fever (CSF) is one of the most severe diseases of pigs, and can cause immense economic losses. Real-time reverse transcription polymerase chain reaction (rRT-PCR) can be used as a sensitive and specific method for detection of Classical swine fever virus (CSFV). Different published protocols are used routinely for CSFV diagnosis. However, almost all these systems use the highly conserved 5′ untranslated region (5′UTR) of the CSFV genome as template.For a reliable diagnosis in outbreaks, a confirmatory assay that amplifies a different genome region is advisable. In this study a new CSFV specific rRT-PCR system using the NS5A region as template is described. The assay is multiplexed with a β-actin detection system that is used as an internal control in a single tube assay. The system was validated using recent European CSFV field isolates, dilution series of an in vitro transcribed RNA standard, and a panel of RNAs representing all available Pestivirus species and genotypes.It was shown that the new assay allows reliable detection of CSFV genomes independent of the 5′UTR region. It presents a very useful diagnostic tool, also allowing a ‘double check’ approach to rule out 5′UTR amplicon contaminations.