麻疹病毒核酸等温扩增试纸条快速检测技术的研究与应用

发布时间：2018-01-01 14:14

本文关键词：麻疹病毒核酸等温扩增试纸条快速检测技术的研究与应用　出处：《浙江理工大学》2016年硕士论文　论文类型：学位论文

【摘要】：麻疹是由麻疹病毒引起的以发热、出疹为特征的急性呼吸道传染病,该病传染性强,致死率高,是目前导致发展中国家儿童死亡的重要原因。20世纪60年代以来,麻疹疫苗开始广泛使用,使麻疹的发病率、死亡率大幅度降低,但据世界卫生组织(World Health Organization,WHO)统计,全球每年仍有超过2千万麻疹病例。WHO制定的全球控制与消除麻疹计划中,麻疹的实验室检测是麻疹监测的重要组成部分。本研究将逆转录环介导等温扩增技术(Reverse-transcriptase loop-mediated isothermal amplification,RT-LAMP)与带防污染装置的横向流动试纸条检测技术(lateral flow dipstick,LFD)相结合,建立了一种麻疹病毒核酸快速检测的新方法。研究针对麻疹病毒血凝素基因设计6条RT-LAMP扩增引物与1条特异性检测探针,其中上游内引物与探针的5’端分别标记生物素(biotin)与异硫氰酸荧光素(Fluorescein isothiocyanate,FITC),进行带biotin标记的RT-LAMP反应,同时进行与FITC-探针的杂交,最后利用LFD对等温扩增产物完成可视化核酸试纸条检测。通过对检测体系的优化,建立的麻疹病毒RT-LAMP-LFD快速检测技术能在1 h内完成对样本的检测(包括核酸提取),方法检测敏感性与荧光RT-PCR方法相同,达到10个RNA copies/μL,同时与其他常见呼吸道传染病病毒无交叉反应,具有良好的检测特异性。收集494份疑似麻疹患者咽拭子标本,用本研究建立的RT-LAMP-LFD快速方法进行检测,同时与荧光RT-PCR法进行比较,结果显示两种方法对494份临床样本的检测结果无显著性差异。结果表明,本研究研发的麻疹病毒核酸RT-LAMP-LFD检测技术对疑似麻疹患者临床样本的检测速度快,而且灵敏度高、特异性好、操作简单、仪器依赖程度低,有望发展成为麻疹病毒核酸快速检测的有效手段。
[Abstract]:Measles is an acute respiratory infectious disease caused by measles virus, characterized by fever and rash. It is an important cause of child mortality in developing countries. Since 60s of the 20th century, measles vaccine has been widely used, which has greatly reduced the incidence and mortality of measles. But according to WHO World Health Organization. There are still more than 20 million measles cases in the world every year. Laboratory detection of measles is an important part of measles surveillance. Reverse-transcriptase loop-mediated isothermal amplification. RT-LAMP) was combined with the lateral flow stickle-LFDs with anti-pollution devices. A new method for rapid detection of measles virus nucleic acid was established. Six RT-LAMP amplified primers and one specific probe were designed for measles virus hemagglutinin gene. The upstream primer and probe were labeled with biotin and Fluorescein isothiocyanate, respectively. Biotin labeled RT-LAMP reaction and hybridization with FITC- probe were performed. Finally, LFD was used to detect the isothermal products of nucleic acid strips. The detection system was optimized. The established rapid detection technique for measles virus RT-LAMP-LFD can complete the detection of samples within 1 h (including nucleic acid extraction). The sensitivity of the method is the same as that of fluorescent RT-PCR method. It reached 10 RNA copias / 渭 L, and had no cross reaction with other common respiratory infectious diseases virus, and had good detection specificity. 494 specimens of throat swabs of suspected measles patients were collected. The rapid RT-LAMP-LFD method was established in this study and compared with the fluorescent RT-PCR method. The results showed that there was no significant difference between the two methods in 494 clinical samples. The measles virus nucleic acid RT-LAMP-LFD detection technology developed in this study for suspected measles patients clinical samples detection speed, and high sensitivity, specificity, simple operation, low degree of instrument dependence. It is expected to be an effective method for rapid detection of measles virus nucleic acid.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R440;R511.1

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