Abstract:
A highly sensitive and specific real-time PCR method was developed for the reliable and rapid detection of African swine
fever virus (ASFV). The method uses a commercial Universal Probe Library (UPL) probe combined with a spe-cifically
designed primer set to amplify an ASFV DNA fragment within the VP72 coding genome region. The detection range of the
optimized UPL PCR technique was confirmed by analysis of a large panel (n = 46) of ASFV isolates, belonging to 19 of the
22 viral p72 genotypes described. No amplification signal was observed when closely clinically related viruses, such as
classical swine fever, or other porcine pathogens were tested by this assay. The detection limit of the UPL PCR method was
established below 18 DNA copies. Validation experi-ments using an extensive collection of field porcine and tick samples (n
= 260), coming from Eastern and Western African regions affected by ASF, demon-strated that the UPL PCR technique was
able to detect over 10% more positive samples than the real-time TaqMan PCR test recommended in the OIE manual,
confirming its superior diagnostic sensitivity. Clinical material collected during experimental infections with different ASFV
p72 genotypes was useful for assur-ing both the capacity of the UPL PCR for an early viral DNA detection and the
competence of the technique to be applied in any ASF diagnostic target sample. The reliability and robustness of the UPL
PCR was finally verified with a panel of ASFV-infected clinical samples which was repeatedly tested at different times.
Additionally, an internal control PCR assay was also developed and standard-ized using UPL probes within the endogenous
b-actin gene. Finally, the com-plete study offers a new validated real-time PCR technique, by means of a standardized
commercial probe, providing a simple, rapid and affordable test, which is ready for application in the routine diagnosis of
ASF.
