Canine distemper virus (CDV) (Canine Morbillivirus) belongs to the genus Morbillivirus and family Paramyxoviridae (International Committee on Taxonomy of Viruses ICTV, 2019). CDV virions are pleomorphic, ∼ 150 nanometers (nm) in diameter, composed by a helical nucleocapsid surrounded by a lipoproteic envelope. The genome is a linear single-stranded, negative-sense RNA molecule of 15.690 nucleotides (nt). The RNA genome contains six transcriptional units, which encode eight viral proteins: nucleocapsid (N), phosphoprotein (P), V, C, matrix (M), fusion (F), hemagglutinin (H) and large (L) proteins (Sidhu et al., 1993, Nguyen et al., 2016).

CDV infection is widespread in domestic dogs and occasionally affects wild animal species, often resulting in systemic and fatal illness, ranging from respiratory, gastrointestinal, dermatological to neurological signs (Harder and Osterhaus, 1997, Martella et al., 2008, Rendon-Marin et al., 2019, Bodgener et al., 2023). Although antiviral candidates have demonstrated anti-CDV activity, no antivirals have been licensed for canine distemper to date. Consequently, animal treatment is based on supportive care (Carvalho et al., 2013, Xue et al., 2019, Shrestha et al., 2021, Martella et al., 2008, Loots et al., 2017). In this context, early and reliable diagnosis of CDV infection is imperative for managing infected animals and to prevent transmission and outbreaks, in special for high-density environments and for endangered wild animal species (Willi et al., 2015, Wang et al., 2017).

Laboratory diagnosis of canine distemper may be performed by a number of tests. Rapid tests are available in veterinary clinics and hospitals, offering quick and reliable results (An et al., 2008). Immunofluorescence assays are a sensitive alternative for CDV detection in diagnostic laboratories (Athanasiou et al., 2018). Molecular diagnostic based on reverse-transcriptase polymerase chain reaction (RT-PCR) has also been widely used, representing a highly sensitive and specific assay for CDV detection (Frisk et al., 1999, Elia et al., 2006, Geiselhardt et al., 2022).

Several CDV-targeted RT-PCRs have been published, including end-point (Frisk et al., 1999, Rzezutka and Mizak, 2002) and TaqMan-based real-time reactions (RT-qPCR) (Elia et al., 2006, Scagliarini et al., 2007). Point-of-care approach, using portable thermal cyclers (Brown et al., 2020), or double-checking strategy with TaqMan-based RT-qPCR have been described as well (Halecker et al., 2021). Despite the success of these assays, some aspects may be improved. For instance, most primers used for CDV detection were designed based on a limited number of sequences or with little information about the dataset used for their design (Frisk et al., 1999, Elia et al., 2006, Scagliarini et al., 2007). This is an important issue when considering the high CDV genetic diversity (Duque-Valencia et al., 2019). Furthermore, although RT-qPCR reactions have advantages over end-point assays, many laboratories do not have access to real-time platforms - a context that should be remembered and handled appropriately.

Considering the above, we designed a novel primer set for CDV detection by both end-point and real-time assays. Reactions using our primers had a limit of detection (LoD) similar to one of the most commonly used primers for CDV detection (Frisk et al., 1999). Moreover, our primers had higher coverage than that described by Frisk et al. (1999), which may contribute to a better diagnostic performance. Overall, RT-PCR and RT-qPCR assays based on our primers represent an attractive alternative for molecular diagnosis of CDV infections.