Figure 4

Differential diagnosis of natural canine distemper virus infection by multiplex ARMS-PCR. (A) Results of a multiplex PCR using the two primer pairs: F-vacc/R-vacc and F-wt/R-wt. As indicated by the arrowheads, a 590 bp product corresponding to vaccine tempalte was specifically amplified from cDNA of Vacc-P, Vacc-Q and Vacc-N (lane 1 to 3); the 300 bp product was only amplified from the local strains (lanes 4-10). Note: Bands with a higher molecular weight, indicated with an arrow, were products amplifed by the outer primer set, F-vacc and R-wt. (B) Characterisation of CDV strains by the two sets of genotype specific primers in combination with various templates, namely Vacc-P (lane 1), Vacc-Q (lane 2), a local strain (lane 3), Vacc-P and a local strain (lane 4), Vacc-Q and a local strain (lane 5) and a negative control without template (lane 6). As indicated with arrowheads, the amplicons corresponding to a specific template, the vaccine strains (590 bp) and the local strains (300 bp), can be differentiated. (C) RFLP analysis of CDV vaccine strains. A unique Bam H I recognition site was found in Vacc-P and CDV isolates in America lineage, but not in Vacc-N, Vacc-Q and other CDVs in Vaccine lineage. As shown in the lower panel, digestion of Vacc-P PCR product with Bam H I enzyme resulted in a smaller DNA fragment (~500 bp; lane 1), whereas DNA obtained from Vacc-Q and Vacc-N remained intact (590 bp; lane 2 and 3).