马尔堡病毒LAMP可视化快速检测方法的建立

The establishment of a method for rapid visual detection of the Marburg virus using a loop-mediated isothermal amplification assay

目的建立马尔堡病毒的环介导等温扩增（loop-mediated isothermal amplification,LAMP）可视化快速检测方法。方法人工合成马尔堡病毒保守的核蛋白（Nucleoprotein,NP）编码基因作为阳性标准品,设计LAMP扩增引物,以HNB作为可视化指示剂,通过LAMP浊度仪测定方法的灵敏度和特异性。并与常规PCR、实时荧光定量PCR作比较。结果建立的LAMP方法的灵敏度为10^0拷贝/μl,高于常规PCR及实时荧光定量PCR的灵敏度10^3拷贝/μl和10^2拷贝/μl,方法的特异性好,仅马尔堡病毒有扩增曲线且HNB指示剂颜色发生阳性改变,其他病毒扩增均为阴性。体系的扩增效率较高,试验仅需30min。结论建立的HNB-LAMP检测方法简便、快速、灵敏、特异,适用于马尔堡病毒的可视化快速检测。

Objective To use loop-mediated isothermal amplification（LAMP）technology and hydroxynaphthol blue to establish a method for rapid visual detection of the Marburg virus. Methods Genes encoding the conserved nucleoprotein of the Marburg virus served as a positive standard template.Primers were designed and hydroxynaphthol blue was used as an indicator in LAMP.A Loopamp Realtime Turbidimeter was used to monitor amplification in real time and findings were recombined with the results of conventional PCR and real-time quantitative PCR to verify the specificity and sensitivity of this method. Results Results indicated that the LAMP test had a detection limit 100copies/μl while the sensitivity of conventional PCR was 10~3copies/μl and the sensitivity of real-time quantitative PCR was 10~2 copies/μl.Results indicated that LAMP detection was more sensitive than the other methods.The LAMP test had a high specificity.Only the tube with the Marburg virus produced an amplification curve and changed color.The LAMP test did not amplify other viruses,it had a higher amplification efficiency,and it quickly yielded results（30min）. Conclusion The HNB-LAMP method for rapid visual detection as established here had a high level of sensitivity and specificity.This method was useful in the rapid visualization of the Marburg virus.