Development and application of multiplex PCR assays for detection of virus-induced respiratory disease complex in dogs

PIEWBANG, Chutchai; RUNGSIPIPAT, Anudep; POOVORAWAN, Yong; TECHANGAMSUWAN, Somporn

Specific primers for viruses causing CIRDC: The sequences of the primers used for CAdV-2 (E3 gene), CDV (NP gene), CIV (M gene), CPIV (NP gene), CRCoV (S gene) and CaHV-1 (GB gene) amplification were retrieved from previous studies [4, 6, 14, 16] and are shown in Table 1 . In order to ascertain the sensitivity, specificity and interaction of those primers, more than 45 sequences of each target gene were compared by multiple alignments using BioEdit Sequence Alignment Editor Version 7.1.3.0 (Ibis Biosciences, Carlsbad, CA, U.S.A.). The in silico specificity test was performed to select the conserved regions using BLASTn analysis in order to ensure the primer specificity without cross amplification of canine genes. Degenerate primers for CIV, CDV, CAdV-2 and CRCoV were applied (Table 1) . Moreover, the canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control as reported previously [20] .