摘要
根据Ｗｅｓｅｌｉｎｇ发表的犬冠状病毒（ＣＣＶ）Ｋ３７８株纤突蛋白（Ｓ）基因序列，设计合成了４条寡聚核苷酸引物Ｐ１（１８ｂｐ，１５２０～１５３７ｂｐ）、Ｐ２（１９ｂｐ，２０７２～２０９０ｂｐ）和Ｐ３（２０ｂｐ，２６２１～２６４０ｂｐ）、Ｐ４（２０ｂｐ，２９４４～２９６３ｂｐ）。以此为引物，以从美国引进的ＣＣＶＮＬ－１８参考株反转录产物为模板，在国内首先建立了ＣＣＶＲＴ－ＰＣＲ方法，并初步应用于国内ＣＣＶ分离株的鉴定。结果表明，以Ｐ１、Ｐ２和Ｐ３、Ｐ４为引物，只能从ＣＣＶＮＬ－１８株反转录产物中扩增出大小分别为５７１ｂｐ和３４３ｂｐ核苷酸片段，与理论设计值大小一致，而正常ＣＲＦＫ细胞和狂犬病等５种对照病毒扩增结果为阴性。ＲＴ－ＰＣＲ可检出１μＬ做１０５和１０３倍稀释的ＣＣＶＮＬ－１８株反转录产物，说明其具有很高的敏感性。初步应用试验结果，可从２株国内分离的ＣＣＶ反转录产物中扩增出５７１ｂｐ和３４３ｂｐ核苷酸片段。通过本研究不仅建立了敏感、特异的ＣＣＶＲＴ－ＰＣＲ检测方法，也为其Ｓ基因的克隆和序列测定奠定了基础。
A RTPCR method for detecting canine coronavirus (CCV) was established with 2 pairs of primer(P1,P2 and P3,P4) designed and synthesized according to the sequenceof the spike(s) gene of CCV and reverse transcription template of the prototypeof CCV NL18 strain and was used to identify CCV YS1 and CCV CI1 isolated in China.The result showed that the method was specific and sensitive. 571bp and 343bp gene sequences which are the same lenth with designation were amplified withthe 2 pairs of primer from the reverse transcription templates of CCV NL-18,CCV YS1 and CCV CI1 strains. The study not only established a specific and sensitive RT-PCR method to detect CCV but also provided a basis for cloning and sequence analysis to the main domain of CCV S gene.
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
1997年第4期78-84,共7页
Journal of Jilin Agricultural University
基金
解放军"九.五"医药卫生科研基金
