Comparison of one-step RT-PCR and a nested PCR for the detection of canine distemper virus in clinical samples.
OBJECTIVE
To develop a rapid and sensitive method for the detection of canine distemper virus (CDV) by nested PCR using clinical specimens.


METHODS
A nested PCR was developed, compared to a one-step RT-PCR and validated.


METHODS
Two 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.


CONCLUSIONS
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 antemortem diagnosis of CDV infections in dogs.
Shin
Y J
YJ
Department of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea.
Cho
K O
KO
Cho
H S
HS
Kang
S K
SK
Kim
H J
HJ
Kim
Y H
YH
Park
H S
HS
Park
N Y
NY
D000818:Animals; D017931:DNA Primers; D004216:Distemper / Q000175:diagnosis*; D004217:Distemper Virus, Canine / Q000235:genetics / Q000302:isolation & purification*; D004285:Dogs; D005260:Female; D008297:Male; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary
D017931:DNA Primers