【作者】 何群；

【导师】 张学；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2005， 博士

【摘要】 前言 乙肝病毒(HBV)的感染呈世界性的分布,乙肝病毒基因组分为多个亚型,一种是根据S区的氨基酸差异划分的血清型,一种是根据核苷酸序列差异划分的基因型。目前对乙肝基因分型方面的研究表明:HBV的临床特点、预后及治疗结果与HBV的基因型有关,与血清型的关系不明显,所以HBV的基因分型对于临床有重要的指导意义,越来越受到重视。目前临床上对乙肝病毒检测的方法基本是ELISA方法,对乙肝病毒的基因分型通常采用测序的方法,HBV分型试剂正在研究和开发中。 基因芯片的问世为HBV的分型检测提供了一个较好的解决方法,本课题目的在于建立用于HBV病毒检测和分型基因芯片的探针设计和芯片制备方法,建立一个比较完整的基因芯片点样、封闭、杂交和检测的操作体系,并对芯片的特异性、灵敏度和重复性进行测试和分析,对HBV芯片在临床上的应用进行了探索。 实验材料 芯片引物和探针,由上海生工公司合成。 PCR试剂盒,购于华美公司。 临床血清标本来自中国医科大学第一和第二临床附属医院。 仪器:芯片扫描仪(Genomic Solution,Inc),生物芯片点样仪MicroGridⅡ(BioRobotics Lid.),PCR扩增仪(Biometra Personal PCR system,Germany),稳压稳流电泳仪(BIO-RAD MiniⅡ,USA),低温离心机(SIGMA,D-37520,Germany),数显电热恒温干燥箱(202-0,上海阳光实验仪器有限公司)更多还原

【Abstract】 IntroductionHepatitis B virus (HBV) infection is a major cause of liver disease around the world. HBV genome can be divided into several subtypes, among which one is serologic subtype according to the amino acids sequence in S protein of HBV and another is genetic subtype according to the nucleotides sequence. The genotypes of HBV are correlated with HBV clinical syndromes and therapeutic efficacy, so the genotyping of HBV had become more and more important. In traditional clinic the genotype of HBV was confirmed by ELISA method, the new genotyping products are being studied.Genechip provides a new promising means for HBV genotyping. In this study we want to establish a integrated method genechip design for HBV examination , including probe design, slide decoration and manipulation, and do some test and analysis about the specificity and sensitivity of the chips and explore its clinical application.Materials and MethodsMaterials and equipments;Primers and probes of the genechips were synthesized by Shanghai Sangon Co. PCR checkbox was purchased from Huamei Co: Clinical sera were offered by the first affiliated Hospital of China Medical University (CMU) and the second affiliated Hospital of CMU. Genechip scanner was purchased from Genomic Solution, Inc; MicroGrid I was purchased from BioRobotics Ltd. ; PCR system was Biometra Personal PCR system, Germany. Electrophoresis Mini II systemwas purchased from BIO - RAD, USA; Centrifugal was purchase from Sigma, Germany.Methods:1. The bioinformatics analysis and PCR primer design of HBV genome. HBV subtype was found in NCBI s Genbank, and the results were align-mented with ClustalW and their conservative and specific sequences were determined. Oligo6.0 and primer5.0 were used to design genotype probes and PCR primers, the reliability of the probes was analyzed.2. Slide decorationThe effects of different chemical reagents on slide were compared and slide fixation efficiency was detected by fluorescence probe; active chemical groups in slide were protected by acetal reaction.3. The best condition of oligonucleotide probesThe best condition of probe buffer, blocking reagent, and balancing time of the active groups reacted with the probes were tested.4. DNA sample and target sequence obtaining.DNA samples were obtained by different method and amplified with PCR, and the best method was determined. Asymmetrical amplifications were designed to find out best ratio of PCR primers.5. Veracity and specification of HybridizationRandom positive PCR products were sent for sequence analysis, and analysis results were compared with hybridization to test the veracity and specification of the genechips.6. Detection of clinical hepatitis B virus by DNA microarray.We have tested 360 serum samples that were collected from patients in the first Hospital of China Medical University ( CMU) and the second Hospital of CMU with our microarrays. We want to find if there is difference between the two methods by comparing the results obtained from genechips with those from ELISA. The sequence was analyzed and HBV subtypes were determined in NCBI by blast.7. The repetition of genechips test.50 random samples were tested and 3 repeat tests were done for each sam-pie and the results were analyzed.Results1. HBV genome bioinformatics analysis and chip probe and PCR primer design.We have found 40 HBV subtypes genome in Genbank, including 8 genotypes: A, B, C, D, E, F, G and H, which are classified by all the serotypes. Phylogenetic tree and the evolutionary relationships were analyzed and found that C subtype is not only a chain of the phylogenetic tree itself and is also distributed in the subtrees of H and G subtypes . PCR primers and 19 probes were designed according to the difference of the sequences and the conservative consequences at HBV S gene region. The product of PCR was 432bp. Blasting the probes in NCBIs Genbank and analyzing the first 50 sequences of the results, we found the accuracy of completely matching with our designed probes is 96%.2. Slide decorationWe analyzed the stability and signal intensity of decorated chip slide and found the selected TM552 is better than A1100 and A1200. The slides made by our lab, of which the intensity is two times stronger, are better than those purchased. The slides can be preserved longer when the chemical group in slide can be protected by methanol.3. The best condition of oligonucleotide probes.We found that when the probe buffer is 0.2M ,pH =9.0 and the concentration of probe is IOOjxM the signal of hybridization is good. The fixation reaction will be balanced when hydrating time is over 4 days. NaBH4 is better than BSA and dimethylamine as a blocking reagent.4. DNA sample and target sequence obtaining.No distinct difference in 3 methods in DNA sample obtaining was found. We selected the boiling method. When ratio of primers is 1:10, the signal of hybridization is best. The best hybridization temperature is 50^ , the best hybridization time is lh.5. Veracity and specification of Hybridization更多还原