实时荧光RPA快速检测犬冠状病毒方法的建立

Establishment of Real-time Recombinase Polymerase Amplification Assay for Rapid Detection of Canine Coronavirus

为提升口岸进出境犬猫科动物检疫工作效率,针对犬冠状病毒(CCV)基因保守序列,设计特异性引物和荧光探针,建立了CCV实时荧光重组聚合酶等温扩增方法(real-time RPA)。结果表明,该方法具有良好的特异性,与其他犬病毒和同属的冠状病毒均未出现交叉反应;其敏感性高于传统的RT-PCR方法;将该方法应用于CCV感染犬的临床组织样本、体表样本及不同时期和地区分离样本的检测,证实该实时荧光RPA方法具有良好的稳定性和可靠性。与现有的核酸检测方法相比,该实时荧光RPA检测方法在核酸上样20 min内即可判读结果,可满足犬猫科动物CCV快速检疫的需要。

In order to accelerate inspection efficiency,real-time recombinase polymerase amplification(real-time RPA)was developed by using pair of special primers and probe against conserved sequence of canine coronavirus(CCV).The results showed that the new method had good specificity of CCV and had no response to other canine virus and coronavirus.And the new method had higher sensitivity than RT-PCR method.The excellent stability of the new method was confirmed by the detection of CCV infected tissue and clinical samples collected at different time and area.Compared with other detection methods of nucleic acid,the real-time RPA could finish detection within 20 min after nucleic acid isolated and meet the requirement of rapid detection of CCV among canid and felid.