Application of N-PCR for Diagnosis of Distemper in Dogs and Fur Animals

The immunofluorescence test, routinely used for laboratory diagnosis of canine distemper virus (CDV) in Poland, is not sufficiently sensitive and specific. Therefore, the application of reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR (N-PCR) and Southern blot hybridization for detection of phosphoprotein (P) gene of CDV in peripheral blood mononuclear cells (PBMCs) or internal organs of dogs and fur animals was the aim of these studies. The optimal parameters for two-step PCR were elaborated for reference strains of distemper virus and used for testing biological samples collected from dogs, foxes, ferret and mink with spontaneous distemper. PCR product of 1069bp was obtained in one out of 10 dog blood samples, three out of 14 homogenates of internal organs of dogs and one out of five homogenates of internal organs of fox. Reamplification with the use of CDVa and CDVb primers demonstrated the 429bp fragment in six samples, negative by PCR: two samples collected from dogs, two from foxes, one from mink and one from ferret. The specificity of N-PCR was confirmed by Southern blot hybridization. We conclude that two-step PCR is sensitive and specific method for diagnosis of CDV infection.