马尔堡病毒LAMP可视化快速检测方法的建立

发布时间：2018-10-18 10:02

【摘要】：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100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。
[Abstract]:Objective to establish a rapid and visualized method for the detection of Marburg virus by ring-mediated isothermal amplification (loop-mediated isothermal amplification,LAMP). Methods the conserved nucleoprotein (Nucleoprotein,NP) coding gene of Marburg virus was synthesized as positive standard. LAMP primers were designed and HNB was used as visual indicator. The sensitivity and specificity of the method were determined by LAMP turbidimetry. And compared with conventional PCR, real-time fluorescence quantitative PCR. Results the sensitivity of the established LAMP method was 100 copies / 渭 l, which was higher than that of conventional PCR and real-time fluorescence quantitative PCR. The sensitivity of the method was 103 copies / 渭 l and 102 copies / 渭 l respectively. The specificity of the method was good. Only Marburg virus had amplification curve and the color of HNB indicator had positive change. All other virus amplification was negative. The amplification efficiency of the system was high, and the experiment only needed 30 minutes. Conclusion the established HNB-LAMP detection method is simple, rapid, sensitive, specific and suitable for rapid visual detection of Marburg virus.
【作者单位】：中国药科大学生命科学与技术学院;南京军区军事医学研究所;
【基金】：国家重大传染病防治专项(No.2013ZX10004103,2013ZX10004218) 江苏省科技支撑计划(社会发展)项目(No.BE2013603) 军队重点项目(No.BWS14J025)
【分类号】：R373;R440

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