新型冠状病毒实验室诊断方法学研究

【作者】 李振勇；

【导师】 赵玉芬；

【作者基本信息】 郑州大学 ， 有机化学， 2005， 博士

【摘要】 2002年11月,中国广东省出现了首例传染性非典型肺炎患者,世界卫生组织根据临床症状于2003年3月15日将其命名为严重急性呼吸道综合症——Severe Acute Respiratory Syndrome(SARS),并于4月16日根据多国协作研究成果,正式宣布SARS的病原体为一种变异的新型冠状病毒(正单链RNA病毒,SARS CoV)。其后,WHO公布的资料显示,全球共计出现非典病人和疑似病人8450例,其中死亡784人,涉及33个国家和地区。由于其病征与感冒、腹泻等常见病病征相似,容易造成临床上的疑诊和误诊。因此,开展相关检测方法和试剂的研发,提供对SARS的早期诊断结果,是进行疾病预防、阻断传播、保护人类自身的首要措施之一,是世界性医学和社会的重大课题。 近两年的研究表明,SARS CoV S蛋白的受体已经很清楚(ACE2),是病毒的主要保护性抗原,N蛋白和M蛋白也可以诱导免疫效应,是检测病毒抗体的良好抗原。本研究根据基因扩增、克隆表达和酶联免疫技术,结合生物信息学、F(?)ster等理论,以基因工程、荧光探针、高效核酸杂交等先进技术手段,提出了简单二级结构高熵值靶序列、空间压缩荧光/淬灭、反转录/抗污染整合等新思路,克服了荧光探针背景过高、RNA提取效率低、阳性对照具有潜在传染性/不稳定、反转录和抗污染不能兼顾等技术难题,取得了特异、适合检测的靶序列和引物、TaqMan-BC探针、高效提取方法、病毒样颗粒阳性对照和内对照、反转录/UNG系统抗污染、cDNA富集等技术创新,建立了荧光RT-PCR基因检测、酶免RT-PCR基因检测、酶免抗体检测等方法和试剂,是SARS早期鉴别诊断、病程监测、预后和流病调查的重要手段,社会意义巨大。 一、根据生物信息学理论,将核酸序列简单二级结构新思路引入靶序列的选择上。并根据基因组信息分析结果,获得了适合基因检测的高熵值靶序列;成功地设计了独特的TaqMan-BC荧光探针,将实际检测本底降低2倍以上; 二、建立了高效简便的核酸提取方法,巧妙设计了反转录/UNG抗污染、cDNA富集、扩增一体化组合的反应体系,在有效提高灵敏度的同时彻底解决了PCR实验过程中容易出现的污染问题,检测灵敏度达到45Copies/mL。 三、整合了RT-PCR和快速核酸杂交技术,通过化学交连,将探针高效地固定在更多还原

【Abstract】 The initial case of Severe Acute Respiratory Syndrome (SARS) was found in 11 November 2002 in Guangdong Province, South China. The disease spread throughout China and to other areas of the world rapidly. According to WHO, there was a cumulative number of 8450 cases with 727 deaths in 33 countries and regions globally during the SARS epidemic. After a more though and comprehensive research carried out by the multi-laboratory international network, WHO officially announced that this new coronavirus, provisionally termed SARS-associated coronavirus (SARS CoV), was the causal agent of SARS.SARS CoV is a newly identified member of the Coronoviridae family of enveloped, positive-stranded RNA viruses. Four putative structural genes, i.e. spike protein(S), envelop protein (E), membrane protein (M) and nucleocapsid protein (N) could be identified. Research in the past year has revealed that S, N, and M protein can induce immunogenicity. The N protein as an immunogen in particular is now believed to be the best choice for antibody-oriented diagnostic assays. The interaction between S protein and tissue-specific host cell receptors (ACE2) trigger fusion of viral envelop with cellular plasma or endosomal membrane, followed by a series of events relating to virus infection and host cell response.At present, no clearly defined treatment regimen and vaccines for SARS are available. The clinical presentation of SRAS is unspecific, resembling the symptoms of other forms of "atypical pneumonia". In this context, early and rapid identification of cases followed by appropriate management such as isolation of suspect and probable cases and management of their close contacts is the only effective way for the control of SARS spread. Suspect and probable cases of SARS should be subjected to laboratory testing for exclusion or confirmation of SARS infection. The availability of reliable and highly specific laboratory tests is of critical importance in the early and rapid diagnosis of SARS.This study integrated many up-to-date technologies such as PCR, in vitro cloning, ELISA, and fluorescence detection. Several original ideas such as "simple secondary structure" for the selection of target nucleotide sequence, "spatially compressed arrangement" for efficient FRET, and "anti-contamination reverse transcriptionsystem" were raised and applied in the design of the experiments, leading to 6 great innovations, including the obtaining of highly specific primers for SARS CoV, successful design of TaqMan-BC fluorescent probes, establishment of a highly efficient method for viral extraction, construction of pseudo-viral particles, introduction of RNase resistant internal control, and the establishment of RT/UNG anti-contamination system. Quantitative Fluorescent RT-PCR System for SARS CoV Detection, ELISA RT-PCR System for SARS Assay, and Double-antigen Sandwich ELISA Assay for SARS Diagnosis released from this study were licensed by SFDA. The main points of the results from this study are summarized below:1. With the idea of evaluating the simple secondary structure of nucleotide sequence, a novel TaqMan-BC fluorescence probe was invented and successfully applied in the fluorescence detection system, which can reduce the detection background by over 2 fold.2. An original RT/UNG combination was successfully established which can effectively eliminate contaminations which are frequently encountered in RT-PCR operations.3. A rapid and sensitive assay combining RT-PCR with rapid nucleotide hybridization was established.4. We are the first in the world that utilized synthesized vrial-like particles to serve as the internal control for SARS CoV detection. By use viral-like particles to monitor the entire detection process, false positive and false negative results could be eliminated. The viral-like particles, unlike inactivated SARS CoV or SARS CoV RNA samples which are liable to degeneration and potentially contagious, but being used in most of the assay systems, are safe, stable, and easy to handle.5. The N protein and M protein of SARS CoV were successfully cloned, expressed, purified, and utilized for the development of the double-antigen sandwich ELISA assay system. The sensitivity, specificity, stability, and reproducibility of the assay system we developed reach the standards for laboratory quality control.6. Monoclonal antibodies against M and N proteins were successfully produced. The proteins produced in vitro which are safe to human can be used as positive controls for the assay process.7. 402 samples from SARS patients and 2106 samples from healthy donors, Non-SARS patients were tested with the double-antigen sandwich ELIS A assay system. The percentage of reactive cases detected with the samples collected at the period of the disease course ( day 10 to 30) by this system is 87%. 2106 samples from healthy donors, Non-SARS patients were tested with this SARS detection system with negative results.8. The percentage of reactive cases detected with the 202 gargling liquid samples collected from SARS patients by PCR system is 56.4%. The other samples from healthy donors, Non-SARS patients were tested with PCR SARS detection system with negative results.Three detection methods for SARS detection have been established.更多还原