Shin Y J, Cho K O, Cho H S, Kang S K, Kim H J, Kim Y H, Park H S, Park N Y
Department of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea.
Aust Vet J. 2004 Jan-Feb;82(1-2):83-6. doi: 10.1111/j.1751-0813.2004.tb14651.x.
To develop a rapid and sensitive method for the detection of canine distemper virus (CDV) by nested PCR using clinical specimens.
A nested PCR was developed, compared to a one-step RT-PCR and validated.
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.
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.
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.
建立一种利用临床标本通过巢式PCR检测犬瘟热病毒（CDV）的快速灵敏方法。
开发了一种巢式PCR，与一步法RT-PCR进行比较并验证。
从犬瘟热病毒核衣壳蛋白（NP）基因的高度保守区域中分别选择两组用于一步法RT-PCR和巢式PCR的特异性引物，分别靶向一个640 bp片段和一个297 bp片段。使用巢式PCR和一步法RT-PCR扩增一株犬瘟热疫苗株的部分犬瘟热病毒NP基因以及29只疑似患犬瘟热的犬的尿液、血液、鼻分泌物和唾液样本。
一步法RT-PCR和巢式PCR均与犬瘟热疫苗株发生反应，但与犬细小病毒不发生反应。一步法RT-PCR在11/22（50.0%）的血液、10/20（50.0%）的尿液、5/25（20.0%）的唾液和6/27（22.2%）的鼻拭子样本中检测到NP基因的预期640 bp片段，而巢式PCR在18/22（81.8%）的血液、15/20（75.0%）的尿液、14/25（56%）的唾液和19/27（70.3%）的鼻拭子样本中扩增出NP基因的预期297 bp片段。
巢式PCR比一步法RT-PCR更频繁地在血液、尿液、鼻拭子和唾液中检测到犬瘟热病毒。因此，该检测方法应有助于犬瘟热病毒感染犬的生前诊断。
