Dirofilaria immitis and Dirofilaria repens are the most common species of filarial nematodes
described in the dogs with increasing spread into new geographical areas. The diagnosis of
canine dirofilariosis is usually based upon the microscopical detection and identification of
circulating microfilariae together with ELISA detection of serum circulating heartworm
antigens or antibodies. The identification of the parasite species using the traditional
approaches sometimes can be difficult and can lead to misdiagnosis especially on samples
from areas where both Dirofilaria are present. In this paper we report a new molecular
method based on single-step multiplex PCR to detect and differentiate simultaneously and
unequivocally D. immitis and D. repens on DNA extracted from canine peripheral blood.
The amplification was performed using a set of primers designed on a portion of the small
subunit ribosomal RNA gene of the mitochondrion (12S rDNA). The single-step multiplex
PCR here described ensured high (4 mf/ml) sensitivity and specificity with reduced cost
and time saving. The multiplex PCR assay represents an additional tool for epidemiological
studies and routine disease assessment in areas co-endemic for the two Dirofilaria species.