第一部分：人类肠道病毒分子生物学分型鉴定方法的建立及应用第二部分：乙型肝炎病毒多表位抗原酵母表达质粒的构建

发布时间：2018-08-27 15:23

【摘要】：人类肠道病毒(HEV)是小核糖核酸病毒科的一个属,文献报道的HEV已有91个型,分为脊髓灰质炎病毒、柯萨奇A组、B组病毒、埃可病毒和新型肠道病毒5个组。人类肠道病毒感染大多表现为隐性感染或症状轻微,但也可以引起无菌性脑膜炎、弛缓性麻痹、急性心肌炎、手足口病等严重疾病。常规的HEV鉴定多采用病毒分离结合中和试验等血清学方法,由于HEV血清型繁多,血清学分型鉴定费时费力。本研究的目的在于建立人类肠道病毒分子生物学分型鉴定方法,用于临床分离肠道病毒的分型鉴定。根据已知各型别人类肠道病毒基因的核苷酸序列和文献资料,分别合成1对HEV种属特异性引物“EV1/EV2”和5对分型鉴定引物“P1/P2”、“P1/P3”、“P4/P7”、“P5/P7”和“P6/P7”,提取病毒RNA,采用逆转录聚合酶链式反应(RT-PCR)扩增病毒cDNA,通过琼脂糖凝胶电泳和核苷酸序列分析,建立了人类肠道病毒的分子生物学分型鉴定方法。采用该方法鉴定了18株疑似HEV毒株,并与血清中和试验相互验证。结果表明,经种属特异性引物鉴定,其中17株为人类肠道病毒;采用型特异性引物进行分型鉴定,其中4株为科萨奇病毒A24型,3株为科萨奇病毒B3型;科萨奇病毒B2、A9、A15型、埃可病毒3、6、7、9、11、14、33型和鼻病毒9型各1株,微量中和试验结果与分子生物学方法分型结果完全符合。本实验组合运用了上述6对HEV鉴定和分型引物,对全部引物的PCR扩增体系和扩增条件进行了统一,可以显著节约病原学检出时间,同时可以确定流行株可能发生的基因变异情况,与传统的病毒分离和血清学鉴定分型方法相比具有特异性强、敏感性高、快速简便等优点,可以直接鉴定并分型诊断人类肠道病毒感染,适用于人类肠道病毒感染的实验室诊断和流行病学研究。
[Abstract]:Human enterovirus (HEV) is a genus of small ribonucleic acid viridae. There are 91 types of HEV reported in the literature, which are divided into five groups: poliovirus, Coxsackie A group B virus, Echo virus and new enterovirus group. The infection of human enterovirus is mostly manifested as recessive infection or mild symptoms, but it can also cause aseptic meningitis, flaccid paralysis, acute myocarditis, hand, foot and mouth disease and other serious diseases. Serological methods such as virus isolation combined with neutralization test and so on are used in routine HEV identification. Because of the variety of serotypes of HEV serological typing is time-consuming and laborious. The purpose of this study was to establish a molecular biological typing method for the identification of human enterovirus. According to the nucleotide sequence and literature data of other enterovirus-like genes, one pair of HEV species-specific primers "EV1/EV2" and five pairs of primers "P1/P2", "P1/P3", "P4/P7", "P5/P7" and "P6/P7" were synthesized respectively. Virus RNA, was extracted by reverse transcription polymerase chain reaction (RT-PCR) amplification of virus cDNA, by agarose gel electrophoresis and nucleotide sequence analysis the molecular biological typing method of human enterovirus was established. Eighteen suspected HEV strains were identified by this method and verified with serum neutralization test. The results showed that 17 of them were human enterovirus by species-specific primer identification, 4 of them were identified by type-specific primers, 4 of them were type B3 of Coxsackievirus A24 and 4 of them were type B3 of Coxsackievirus B _ (2) A _ (9) A _ (15). The results of microneutralization test were in good agreement with the results of molecular biological typing. In this experiment, six pairs of primers for HEV identification and typing were used to unify the PCR amplification system and amplification conditions of all primers, which could significantly save the time of pathogen detection. At the same time, the possible genetic variation of the epidemic strain can be determined. Compared with the traditional virus isolation and serological identification and typing method, it has the advantages of high specificity, high sensitivity, rapid and simple, and so on. Human enterovirus infection can be directly identified and typed. It is suitable for laboratory diagnosis and epidemiological study of human enterovirus infection.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R446.9;R373.21

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第一部分：人类肠道病毒分子生物学分型鉴定方法的建立及应用 第二部分：乙型肝炎病毒多表位抗原酵母表达质粒的构建

第一部分：人类肠道病毒分子生物学分型鉴定方法的建立及应用第二部分：乙型肝炎病毒多表位抗原酵母表达质粒的构建