犬瘟热病毒RT-PCR检测方法的建立与应用

Establishment of CDV RT-PCR Method and Application

根据Barrett发表的犬瘟热病毒 (CDV)Onderstepoort弱毒株融合蛋白 (F)基因序列 ,设计合成了一对寡聚核苷酸引物 ,并以南京农业大学惠赠的Onderstepoort标准株反转录产物为模板 ,建立了CDV的RT PCR方法 ,并应用于CDV的诊断。结果表明 ,以该对引物 ,只能从CDV ONP标准株反转录产物中扩增出大小为 32 4bp的核苷酸片段 ,与理论设计值大小一致 ,而正常Vero细胞和同为副粘病毒科的新城疫病毒 (NDV)作为对照 ,病毒扩增结果为阴性。RT PCR可检出 1μl作 10 6倍稀释的CDV ONP标准株反转录产物 ,说明其具有很高的敏感性。应用试验结果 ,可从感染CDV犬的组织病料中提取RNA ,反转录产物中也扩增出 32 4bp的核苷酸片段。通过本研究不仅建立了敏感性、特异性好的CDV的RT PCR检测方法 ,也为F基因的克隆和序列测定及表达奠定了基础。

A RT-PCR method for detecting canine distemper virus (CDV) was established with one pair of primer(P1、P2) designed and synthesized according to the sequence of the fusion(F) gene of CDV and reverse transcription template of the standard of CDV-ONP strain and was used to identify the isolate. The result showed that the method was specific and sensitive. 324bp gene sequence which is the same length with designation is amplified with one pair of primer from the reverse transcription template of CDV-ONP strain. The study has not only established a specific and sensitive RT-PCR method to detect CDV but also provided a basis for cloning , sequence analysis and expressing to the main domain of CDV F gene.