Kim Y H, Cho K W, Youn H Y, Yoo H S, Han H R
Department of Internal Medicine and Infectious Diseases, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea.
J Vet Sci. 2001 Apr;2(1):59-63.
A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection.
在巢式PCR技术建立之后，为提高犬瘟热病毒（CDV）感染诊断的效率，建立了一步法逆转录PCR（RT-PCR）联合巢式PCR技术。基于CDV Onderstepoort株核衣壳基因序列设计了两套PCR引物。结果显示，使用外引物对进行一步法RT-PCR可检测到10(2) PFU/ml的病毒。将一步法RT-PCR与巢式PCR联合使用，灵敏度提高了100倍。还使用其他相关犬病毒以及健康犬的外周血单个核细胞（PBMC）和身体分泌物对PCR的特异性进行了验证。在51份临床疑似患有犬瘟热的犬血样中，45份样本经一步法RT-PCR联合巢式PCR检测呈阳性。然而，仅15份样本通过单一步法RT-PCR鉴定为阳性。因此，联合方法的诊断效率提高了约60%。这些结果表明，一步法RT-PCR联合巢式PCR可能是一种用于诊断CDV感染的灵敏、特异且实用的方法。
