小鼠仙台病毒LAMP快速检测方法的建立

Establishment of the LAMP rapid detection method for mouse Sendai virus

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 establish a method for the detection of the loop mediated isothermal amplification（LAMP）of mice Sendai virus（Se V）. Methods：Using LAMP technology,six specific primers for LAMP were designed with V4 Primer Exploer software,according to the target gene sequence of mice of Se V（gi：9627219）F protein. Se V RNA was extracted with virus RNA kit. Se V specific LAMP（Se V/LAMP）method was established through optimizing the LAMP reaction system. At the same time,validation of specificity,stability,repeatability and sensitivity of the method,and initially clinical testing were performed. Results：The established method of Se V/LAMP can detect RNA Se V specifically,having no reaction with other common pathogens. Se V/LAMP can detect different batches at the same time,or the same batch at different times of RNA Se V. The lower limit of Se V/LAMP detection of Se V RNA was 0.33 pg;the sensitivity of Se V/LAMP detection of Se V RNA was high（respectively 5/30,2/30）compared with that of ELISA detection method. For clinical detection,the positive rate of Se V was 27.5%（11/40）. All reactions can be completed within 1 h. Visual experimental results can be achieved by adding calcein intuitive. Conclusion ：LAMP method has the advantages of specificity,accuracy,sensitivity,rapid and simple,and therefore can be used for the rapid detection of Se V in mice.