等温核酸扩增技术检测中东呼吸综合征冠状病毒和鼻病毒的研究

发布时间：2018-08-12 12:42

【摘要】：病毒性传染病,尤其是可经呼吸道感染的病毒性传染病,一直是全球公共卫生领域关注的焦点。一方面,以西尼罗病毒,埃博拉病毒为代表的已知病毒重新出现引起疾病暴发；另一方面,以中东呼吸综合征冠状病毒,H7N9禽流感病毒为代表的新发传染病不断出现。大多数病毒性传染病尚无有效的药物和疫苗,因此快速可靠的诊断方法的研发和应用成为预防和控制病毒性传染病的重要措施。目前,病原核酸检测方法大多基于聚合酶链式反应(Polymerase chain reaction, PCR)及其延伸技术,但该法耗时长且需要精密昂贵仪器,不适于在基层检验检疫或医疗卫生机构中使用。相比而言,等温核酸扩增技术,如环介导等温扩增(Loop mediated isothermal amplification, LAMP),基因组指数扩增反应(Genome Exponential Amplification Reaction, GEAR)和等温多自配引发扩增(Isothermal multiple-self-matching-initiated amplification, IMSA)在恒温条件下即可发生扩增,具有快速、高特异性和灵敏度等特点,对硬件设备要求低,更适合基层检测工作的需要。首先,本研究基于上述三种等温扩增技术建立了对中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus, MERS-CoV)这一新发病原体的检测方法。选择MERS-CoV的核衣壳蛋白基因(Nucleocapsid, N)设计引物并在等温条件下(63℃)进行扩增反应。通过实时荧光法和颜色判定法进行检测结果判定。对多种人类冠状病毒及常见呼吸道病毒进行了特异性验证,无交叉反应。对梯度稀释的体外转录MERS-CoV N;基因RNA进行了检测限分析,同时与美国CDC发表的实时荧光定量PCR (real time RT-PCR, rRT-PCR)进行了比较。结果显示,通过实时荧光法判读结果时,RT-LAMP和RT-GEAR检测限相当,为5×102拷贝/反应；RT-IMSA和rRT-PCR相当,为102拷贝/反应。通过颜色判定法判断结果时,RT-IMSA检测限为5×102拷贝/反应,而另外两种等温扩增方法均为103拷贝/反应。对含MERS-CoV N基因扩增区域的病毒样颗粒(Virus-like particles, VLPs)进行检测时,RT-LAMP和RT-GEAR的灵敏度均为104拷贝/反应,而RT-IMSA的灵敏度可达103拷贝/反应,优于上述两种反应。因此,上述等温检测方法有望应用于MERS-CoV感染的快速筛选,具有在基层医疗卫生机构和现场推广和应用的潜力。其次,本研究基于GEAR技术建立了针对鼻病毒(Human rhino viruses, HRVs)的通用型等温扩增检测方法。针对HRVs的5’非翻译区(Untranslated region, UTR)基因设计4条特异引物,在等温条件下(65℃)进行60 min扩增反应,通过Genie(?) Ⅱ等温扩增仪收集荧光信号,实时监测扩增结果。采用常见肠道病毒和呼吸道病毒进行了特异性验证,无交叉反应；采用含HRV-A60、 HRV-B06、HRV-C07扩增区域的体外转录RNA进行了灵敏度评价,灵敏度分别为5,50和5拷贝／反应。通过与文献报道的半巢式PCR后产物测序方法同时检测143份临床标本,评价了该方法的临床检测效果。RT-GEAR的特异性和灵敏度分别为100%和98.08%,kappa值为0.985。结果显示,基于便携式Genie(?)II等温扩增仪的RT-GEAR等温核酸扩增检测方法是一种高效快速,可用于现场的HRVs通用型检测方法。综上所述,本研究成功建立了针对新发病原MERS-CoV的核酸快速等温扩增检测方法,为MERS-CoV感染诊断提供了快速有效手段,为检验检疫和医疗卫生部门的检测工作提供了技术支持,为应对国内可能发生疫情提供了技术储备,并具有在现场应用和向基层推广的潜力。此外,本文也建立了针对流行率最高的人类呼吸道感染病毒HRVs的通用型核酸等温扩增检测方法,可应用于HRVs所有基因型的检测,为HRVs感染诊断提供了普适、可靠和快速的手段,有助于其引起疾病的早期诊断进而防止严重并发症的发生。
[Abstract]:Viral infectious diseases, especially viral infectious diseases that can be transmitted through the respiratory tract, have always been the focus of public health worldwide. On the one hand, known viruses such as West Nile virus and Ebola virus reappear to cause disease outbreaks; on the other hand, Coronavirus of Middle East Respiratory Syndrome, H7N9 avian influenza virus are the representatives. Most viral infectious diseases do not have effective drugs and vaccines, so the development and application of rapid and reliable diagnostic methods become an important measure to prevent and control viral infectious diseases. In contrast, isothermal nucleic acid amplification techniques such as Loop mediated isothermal amplification (LAMP), Genome Exponential Amplification Reaction (Genome Exponential Amplification Reaction) GEAR and Isothermal multiple-self-matching-initiated amplification (IMSA) can be amplified at constant temperature. They have the characteristics of fast, high specificity and sensitivity. They require less hardware and are more suitable for the detection work at the basic level. A method for the detection of a new pathogen, Middle East respiratory syndrome coronavirus (MERS-CoV), was established. The primers of the nucleocapsid gene (N) of MERS-CoV were designed and amplified under isothermal conditions (63 C). Real-time fluorescence and color determination were used. The specificity of several human coronavirus and common respiratory viruses was verified without cross reaction. The detection limits of gradient dilution MERS-CoV N and gene RNA were analyzed and compared with real-time RT-PCR (rRT-PCR) published by CDC. The detection limit of RT-LAMP and RT-GEAR was 5 *102 copies/reactions, and that of RT-IMSA and rRT-PCR was 102 copies/reactions. The detection limit of RT-IMSA was 5 *102 copies/reactions by color determination, while the other two isothermal amplification methods were 103 copies/reactions. The sensitivity of RT-LAMP and RT-GEAR was 104 copies/reactions, while the sensitivity of RT-IMSA was 103 copies/reactions, which was superior to the above two reactions. Therefore, the above isothermal detection method is expected to be applied to the rapid screening of MERS-CoV infection, with basic medical and health institutions and now available. Secondly, a universal isothermal amplification assay for human rhino viruses (HRVs) was developed based on the GEAR technique. Four specific primers were designed for the 5'untranslated region (UTR) gene of HRVs and amplified by Genie (?) II Isothermal amplifier was used to collect fluorescent signals and monitor the amplification results in real time.The specificity of enteroviruses and respiratory viruses was tested without cross-reaction.The sensitivity of in vitro transcriptional RNA containing HRV-A60, HRV-B06 and HRV-C07 amplified regions was evaluated with sensitivity of 5,50 and 5 copies/reaction respectively. The specificity and sensitivity of RT-GEAR were 100% and 98.08% respectively, and the kappa value was 0.985. The results showed that the RT-GEAR isothermal nucleic acid amplification method based on portable Genie (?) II isothermal amplifier was efficient, rapid and available. In summary, a rapid isothermal amplification method for detecting the new pathogen MERS-CoV was successfully established, which provided a rapid and effective means for the diagnosis of MERS-CoV infection, provided technical support for the inspection and quarantine work and the detection work of the medical and health departments, and provided a possible epidemic situation in China. In addition, a universal nucleic acid isothermal amplification assay for the detection of human respiratory tract infection virus (HRVs) with the highest prevalence rate has been established, which can be used for the detection of all genotypes of HRVs and provides a universal, reliable and rapid method for the diagnosis of HRVs infection. It helps to diagnose early diseases and prevent serious complications.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R511;R440

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