小反刍兽疫灭活抗原RT-PCR检测方法的建立及其相关序列分析

Establishment of RT-PCR for Detecting the Inactivated Antigens of Peste des petits ruminants virus and Its Related Sequence Analysis

以灭活的检测用抗原为材料,抽提灭活小反刍兽疫病毒的基因组RNA作为模板,根据参考文献及GenBank下载的序列,设计3对位于F基因的引物(2对为套式引物),进行RT-PCR扩增及扩增片段的T载体克隆、序列分析。结果显示,所设计引物单独或套式RT-PCR对模板均有预期大小的片段扩增;扩增片段经核苷酸序列测定和分析,表明所扩增片段为小反刍兽疫病毒的F基因片段。且所设计引物对同属牛瘟病毒、犬瘟热病毒无扩增,证明所用引物为小反刍兽疫病毒特异性引物,此3对引物可用于小反刍兽疫与牛瘟等同属病毒的鉴别诊断。

In this study, the inactivated PPRV antigen was used as study material. Its genome RNA was ex- tracted as the RT-PCR template. According to references and the downloaded sequences, three sets of primers of F gene were designed. Then RT-PCR amplification was performed and the amplified fragments were cloned to the pMD18-T vector, sequenced and analyzed. The results showed that the desired frag- ments can be amplified by the designed primers whether in the RT-PCR or the nested RT-PCR methods. Through sequencing analysis,the amplified fragments were correct and all sited in the F gene fragments. The primers used in this study are special to PPRV and can＇t amplify the viruses of the same genus, RPV and canine distemper virus（CDV）. The methods can be used to differentiate the alike viruses.