Characterisation of the discrepancy between PCR and virus isolation in relation to classical swine fever virus detection.

In order to confirm and characterise further the discrepancies observed between diagnostic RT-nPCR and virus isolation results for the detection of classical swine fever virus (CSFV), a test panel of three new RT-PCRs was designed, amplifying parts of the NS2, NS3 and NS5A regions. Screening of negative samples by virus isolation with the new panel not only confirmed the discrepancies previously observed but also indicated that these were not associated with a specific genomic region. However, none of the PCR-positive samples were positive on all the different PCRs and preferential amplification was not obtained even when a more sensitive real-time RT-PCR was used. Furthermore, the primer-dependent amplification, most likely caused by the presence of viral fragments, demonstrates the necessity of confirming a single positive PCR result, certainly in the presence of contradictory virus isolation results. The new PCR panel, in combination with sequencing, can be used as a tool to provide additional information on the nature of the viral RNA present in the sample.