Canine Parvovirus and Canine Coronavirus Detection

For detection of CanineCV in dog samples, a conventional PCR assay was performed using a set of specific primers reported by Kotsias et al.^{25 (link)}. Briefly, the primers Forward For-genomic-(5′-ATGGCTCAAGCTCAGGTTG-3′) and the Rev 533 Reverse primer (5′-CCGCACAGAACCTCCACTTC-3′) were used. To confirm CPV-2-positive samples, PCR amplification of VP2 was conducted using the Forward Ext1F primer (5′-ATGAGTGATGGAGCAGTTCA-3′) and the Ext3R Reverse primer (5′-AGGTGCTAGTTGAGATTTTTCATATAC-3′) described by Ref.^{26 (link)}. In all cases, reactions were performed in a total volume of 25 ul containing 1 unit of Taq polymerase (Go taq flexi-Promega), 1 × Taq buffer, 2 µM MgCl₂, 0.5 µM dNTPs, 20 pmol of each primer and 100 ng of extracted DNA. The PCR reactions were performed on a C1000 Touch BIORAD-DNA thermocycler (Biorad; CA, USA). Molecular grade water was used as a negative control for amplifications. PCR amplification results were visualized using 1.5% horizontal agarose gel electrophoresis stained with the Invitrogen SYBR Safe DNA Gel Stain (Thermo Fisher Scientific). The GeneRuler 100-bp DNA Plus Ladder (Thermo Fisher Scientific) was used as a molecular weight marker. Gels were visualized in the ultraviolet light Gel Doc XR + imaging system (Bio-Rad, Molecular imager, USA) by using ImageLab software.