Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set of four primers was designed to amplify six target sequences at the gp48 gene region for the RT-LAMP assay. The optimization of the RT-LAMP reaction was performed by evaluat-ing reaction temperature and reaction time. [Result] The RT-LAMP aasay was suc-cessful y conducted at 56 ℃ within 40 min under isothermal conditions, and the re-sults could be detected as ladder-like bands using agarose gel electrophoresis. The RT-LAMP assay is highly sensitive and able to detect 3.74 ×100 copies/μl of BVDV RNA, as no cross-reaction was observed with other viruses. [Conclusion] Overal , the newly established RT-LAMP assay indicates the potential application in both clinical diagnosis and field surveil ance of BVDV.
根据GenBank中登录的PRRSV Nsp9基因序列,应用Primer Explorer V4软件,在该基因序列中选取保守区设计了4条RT-LAMP引物,旨在建立1种针对Nsp9蛋白基因的逆转录环介导等温扩增(RT-LAMP)的快速检测方法。采用引物分组扩增的方法对引物进行了检测,以确保引物的可靠性。对反应体系、温度以及时间进行了优化,检测了该方法的特异性和灵敏度。结果表明,该方法在等温条件下只需50min就能检测出结果。与RT-PCR方法相比,在判定检测结果时不需要借助昂贵仪器设备,具有高特异性、高灵敏度、操作简便快速等特点,适合于临床检测的应用。
