Comparison of one‐step RT‐PCR and a nested PCR for the detection of canine distemper virus in clinical samples

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Abstract
Objective To develop a rapid and sensitive method for the detection of canine distemper virus (CDV) by nested PCR using clinical specimens. Design A nested PCR was developed, compared to a one‐step RT‐PCR and validated. ProcedureTwo sets of specific primers for a one‐step RT‐PCR and a nested PCR, targeting a 640 bp fragment and a 297 bp fragment, respectively, were selected from the highly conserved region of the nucleocapsid protein (NP) gene of CDV. The nested PCR and the one‐step RT‐PCR were used to amplify a part of the CDV NP gene of a CDV vaccinal strain and samples of urine, blood, nasal discharge and saliva from 29 dogs suspected of suffering CD. Results Both the one‐step RT‐PCR and the nested PCR reacted with the CDV vaccinal strain, but not with canine parvovirus. The expected 640 bp fragment of the NP gene was detected in 11/22 (50.0%) blood, 10/20 (50.0%) urine, 5/25 (20.0%) saliva and 6/27 (22.2%) nasal swab samples by one‐step RT‐PCR, whereas the nested PCR amplified an expected 297 bp fragment of the NP gene in 18/22 (81.8%) blood, 15/20 (75.0%) urine, 14/25 (56%) saliva and 19/27 (70.3%) nasal swab samples. Conclusion The nested PCR detected CDV in blood, urine, nasal swab and saliva more frequently than did the one‐step RT‐PCR. Therefore, this assay should be a useful aid to ante‐mortem diagnosis of CDV infections in dogs.

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