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目的:建立一种实验小鼠仙台病毒(Sendai virus,Se V)的环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法。方法:采用LAMP技术,以小鼠Se V(gi:9627219)F蛋白的基因序列为靶基因,采用Primer Exploer V4软件设计LAMP的6条特异性引物,应用病毒RNA提取试剂盒提取Se V RNA,通过优化LAMP反应体系条件建立小鼠Se V特异性的LAMP检测方法(Se V/LAMP)。同时,对该方法的特异性、稳定性、重复性、灵敏度进行验证,并初步用于临床检测。结果:所建立的Se V/LAMP方法可特异性的检测出Se V RNA,其他常见病原体均为阴性。Se V/LAMP能检测出不同批次同一时间或同一批次不同时间的Se V RNA;Se V/LAMP检测Se V RNA的最低检测含量为0.33 pg,与ELISA检测方法相比较,其灵敏度高(分别为5/30和2/30)。临床检测,Se V的阳性率为27.5%(11/40);全部反应可在1 h内完成。通过添加荧光染料SYBR Green I可直观目测实验结果。结论:建立的LAMP方法,具有特异、准确、灵敏、快速、简便的特点,可用于实验小鼠Se V的快速检测。
Objective: To develop a loop-mediated isothermal amplification (LAMP) assay for Sendai virus (Se V) in mice. Methods: The LAMP gene was used as the target gene of mouse Se V (gi: 9627219) F protein by LAMP technique. Six primers specific to LAMP were designed by Primer Exploer V4 software. The viral RNA extraction kit was used to extract Se V RNA. Mouse Se V-specific LAMP detection method (Se V / LAMP) was established by optimizing the conditions of LAMP reaction system. At the same time, the specificity, stability, repeatability and sensitivity of the method were validated and initially used in clinical testing. Results: The established Se V / LAMP method can detect Se V RNA specifically, and other common pathogens are negative. Se V / LAMP could detect Se V RNA in different batches at the same time or at different times of the same batch. Se V / LAMP assay showed the lowest detection level of Se V RNA was 0.33 pg, which was higher than the ELISA assay 5/30 and 2/30, respectively). Clinical tests, Se V positive rate was 27.5% (11/40); all reactions can be completed within 1 h. By adding fluorescent dye SYBR Green I can visually visualize the experimental results. Conclusion: The established LAMP method is specific, accurate, sensitive, rapid and simple and can be used for the rapid detection of SeV in experimental mice.