【摘要】：病毒性傳染病,尤其是可經(jīng)呼吸道感染的病毒性傳染病,一直是全球公共衛(wèi)生領(lǐng)域關(guān)注的焦點。一方面,以西尼羅病毒,埃博拉病毒為代表的已知病毒重新出現(xiàn)引起疾病暴發(fā)；另一方面,以中東呼吸綜合征冠狀病毒,H7N9禽流感病毒為代表的新發(fā)傳染病不斷出現(xiàn)。大多數(shù)病毒性傳染病尚無有效的藥物和疫苗,因此快速可靠的診斷方法的研發(fā)和應(yīng)用成為預(yù)防和控制病毒性傳染病的重要措施。目前,病原核酸檢測方法大多基于聚合酶鏈式反應(yīng)(Polymerase chain reaction, PCR)及其延伸技術(shù),但該法耗時長且需要精密昂貴儀器,不適于在基層檢驗檢疫或醫(yī)療衛(wèi)生機構(gòu)中使用。相比而言,等溫核酸擴增技術(shù),如環(huán)介導(dǎo)等溫擴增(Loop mediated isothermal amplification, LAMP),基因組指數(shù)擴增反應(yīng)(Genome Exponential Amplification Reaction, GEAR)和等溫多自配引發(fā)擴增(Isothermal multiple-self-matching-initiated amplification, IMSA)在恒溫條件下即可發(fā)生擴增,具有快速、高特異性和靈敏度等特點,對硬件設(shè)備要求低,更適合基層檢測工作的需要。首先,本研究基于上述三種等溫擴增技術(shù)建立了對中東呼吸綜合征冠狀病毒(Middle East respiratory syndrome coronavirus, MERS-CoV)這一新發(fā)病原體的檢測方法。選擇MERS-CoV的核衣殼蛋白基因(Nucleocapsid, N)設(shè)計引物并在等溫條件下(63℃)進行擴增反應(yīng)。通過實時熒光法和顏色判定法進行檢測結(jié)果判定。對多種人類冠狀病毒及常見呼吸道病毒進行了特異性驗證,無交叉反應(yīng)。對梯度稀釋的體外轉(zhuǎn)錄MERS-CoV N;基因RNA進行了檢測限分析,同時與美國CDC發(fā)表的實時熒光定量PCR (real time RT-PCR, rRT-PCR)進行了比較。結(jié)果顯示,通過實時熒光法判讀結(jié)果時,RT-LAMP和RT-GEAR檢測限相當,為5×102拷貝/反應(yīng)；RT-IMSA和rRT-PCR相當,為102拷貝/反應(yīng)。通過顏色判定法判斷結(jié)果時,RT-IMSA檢測限為5×102拷貝/反應(yīng),而另外兩種等溫擴增方法均為103拷貝/反應(yīng)。對含MERS-CoV N基因擴增區(qū)域的病毒樣顆粒(Virus-like particles, VLPs)進行檢測時,RT-LAMP和RT-GEAR的靈敏度均為104拷貝/反應(yīng),而RT-IMSA的靈敏度可達103拷貝/反應(yīng),優(yōu)于上述兩種反應(yīng)。因此,上述等溫檢測方法有望應(yīng)用于MERS-CoV感染的快速篩選,具有在基層醫(yī)療衛(wèi)生機構(gòu)和現(xiàn)場推廣和應(yīng)用的潛力。其次,本研究基于GEAR技術(shù)建立了針對鼻病毒(Human rhino viruses, HRVs)的通用型等溫擴增檢測方法。針對HRVs的5’非翻譯區(qū)(Untranslated region, UTR)基因設(shè)計4條特異引物,在等溫條件下(65℃)進行60 min擴增反應(yīng),通過Genie(?) Ⅱ等溫擴增儀收集熒光信號,實時監(jiān)測擴增結(jié)果。采用常見腸道病毒和呼吸道病毒進行了特異性驗證,無交叉反應(yīng)；采用含HRV-A60、 HRV-B06、HRV-C07擴增區(qū)域的體外轉(zhuǎn)錄RNA進行了靈敏度評價,靈敏度分別為5,50和5拷貝／反應(yīng)。通過與文獻報道的半巢式PCR后產(chǎn)物測序方法同時檢測143份臨床標本,評價了該方法的臨床檢測效果。RT-GEAR的特異性和靈敏度分別為100%和98.08%,kappa值為0.985。結(jié)果顯示,基于便攜式Genie(?)II等溫擴增儀的RT-GEAR等溫核酸擴增檢測方法是一種高效快速,可用于現(xiàn)場的HRVs通用型檢測方法。綜上所述,本研究成功建立了針對新發(fā)病原MERS-CoV的核酸快速等溫擴增檢測方法,為MERS-CoV感染診斷提供了快速有效手段,為檢驗檢疫和醫(yī)療衛(wèi)生部門的檢測工作提供了技術(shù)支持,為應(yīng)對國內(nèi)可能發(fā)生疫情提供了技術(shù)儲備,并具有在現(xiàn)場應(yīng)用和向基層推廣的潛力。此外,本文也建立了針對流行率最高的人類呼吸道感染病毒HRVs的通用型核酸等溫擴增檢測方法,可應(yīng)用于HRVs所有基因型的檢測,為HRVs感染診斷提供了普適、可靠和快速的手段,有助于其引起疾病的早期診斷進而防止嚴重并發(fā)癥的發(fā)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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R511;R440

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