【作者】 郭晓凤；

【导师】 梁伟峰；

【作者基本信息】 浙江大学 ， 临床医学， 2007， 硕士

【摘要】 HBV基因分型检测芯片的制备及临床应用前言乙型肝炎病毒(HBV)的感染呈世界性分布,在我国尤其多见。HBV感染可导致急、慢性肝炎(CHB),甚至肝硬化,肝癌。由于病毒变异,HBV基因组分为多个型。根据其核苷酸序列差异,HBV可划分为(A-H)8种基因型。目前研究认为,HBV基因型是影响HBV感染者预后的重要因素之一,因此建立一套快速、准确的HBV分型检测技术十分必要。目前诊断HBV基因型的常用方法有DNA测序,聚合酶联反应-限制性片段长度多态性分析法(PCR-RFLP)等,在结果准确性、技术简单性方面各有优劣,且不能同时检测所有基因型。近几年发展起来的基因芯片技术弥补上述方法的缺陷。本课题的研究目的在于:建立DNA芯片检测HBV基因分型的研究方法;并对芯片检测的特异性、准确性、灵敏度和重复性进行测试、分析;通过调查杭州地区HBV的基因型分布对HBV基因分型检测芯片在临床上的应用进行初步探索,为病原体基因型检测芯片这种高科技方法在临床上的应用进行积极探索,积累有益经验。方法1.有关探针、引物设计在GenBank数据库中查找HBV各型Pre-S基因组序列,并利用DNAStar软件进行MegAlign分析,找出其保守序列和特征序列。根据生物学软件Primer primer5.0设计出不同分型探针及其PCR引物。并对探针进行分析。2.基因芯片的设计、制作将设计好的多条寡核苷酸探针在醛基基片的特定位置上,用点样仪将探针固定,同一探针平行重复点样3次,以观察其重复性,为了能在每次实验时判定实验操作的正确性,以及是否存在污染,在芯片设计时增加了如下的质控点;标志列、阳性对照行、阴性对照行。3.杂交、显色技术提取血清中DNA,进行PCR扩增后滴于基因芯片上进行杂交,采用酶促显色技术检测杂交结果。4.检测结果的准确性随机抽取PCR阳性标本送交上海生工公司进行测序,测序结果与芯片杂交结果进行对比,观察芯片杂交反应的准确性。5.检测结果的重复性分析取20份HBV-DNA阳性标本,每份标本用本法重复测定3次,观察结果是否一致。结果1.探针、引物设计在Genbank中查到17个HBV Pre-S基因组序列,共为A、B、C、D、E、F、G7个基因型,对其进行了进化树分析和多序列比对。根据序列差异情况和保守序列分布情况设计出可以扩增出特异序列的PCR引物和7个探针,PCR产物长度为340bp左右。到NCBI上将设计好的探针进行BLAST,分析评分在前50个的序列及其完全匹配序列,与探针完全匹配的序列基因型一致的为97%。2.临床标本检测用制备的基因芯片检测HBV各基因型(A-G)标准病毒株DNA样本,检测结果与已知基因型(经核酸测序)完全一致;106例HBV-DNA阳性标本均测出其基因型,其中B型37例,占34.91%,C型69例,占65.09%。未发现其他基因型与混合型感染。3.DNA序列分析测序结果与芯片杂交结果完全一致,进一步证实了检测结果的特异性、准确性。经过测序分析,杂交特异性和探针匹配程度一致。4.检测结果重复性测定和灵敏度分析取20份HBV-DNA阳性标本,每份标本用本法重复测定3次,结果一致,显示出良好的重复性。106例阳性血清,采用荧光定量PCR检测,HBV-DNA水平最低者为2~* 10~4 copies/ml,初步认定该芯片的检测灵敏度不低于此水平。结论1.通过对HBV基因组的生物信息学分析,可以设计出相应的探针和引物用于HBV基因分型芯片制作。2.设计制备的HBV基因分型芯片能检测出HBV基因型,初步应用于临床后,显示出满意的准确性、特异性、稳定性和灵敏度。3.杭州地区HBV感染者HBV基因型B、C两型均存在,但以C型为主,由于选择研究对象不同等因素的存在,其确切分布尚需大样本、多中心临床研究以进一步明确。4.本研究结果表明,基因型检测芯片具有操作简单、自动化程度高、结果准确可靠等特点,有良好的临床应用前景。更多还原

【Abstract】 IntroductionHepatitis B virus(HBV)infection is a major cause of liver disease around the world,especially in China.HBV genome can be divided into several subtypes.According to the nucleotides sequence,HBV has been classified into 8 genotypes(A-H).The genotypes of HBV are correlated with HBV clinical syndromes and therapeutic efficacy,so the genotyping of HBV had become more and more important.Currently there were some diagnostic methods of genotype,including the complete genome sequence analysis or S gene sequencing,PCR-RFLP and so on.But they have the disadvantages of a high cost and time-consuming to carry out or the result lack of reliability,and they haven’t the ability to detect many.targets simultaneously.Recently,as the progress of functional genomic techniques,microarray has been developed into an ideal tool for genotyping.In this study we want to establish a integrated method genechip design for HBV genotyping,and do some test and analysis about the specificity and repetition of the chips and explore its clinical application,And,to investigate the distribution of hepatitis B virus genotypes in Hangzhou area.Methods1.PCR primer and probe design of HBV genome.HBV genotypes were found in NCBI’s Genbank,and the results were alignmented with MegAlign and their conservative and specific sequences were determined.Primer Primer 5.0 was used to design genotype probes and PCR primers,the reliability of probes was analyzed.2.Microarray fabricationThe oligonucleotide probes were stablized on the chip and each one spotted at a layout of 3×1 blocks to avoid possible hybridization failures.Six blocks of the HBV-Con probes targeting the conserved region of Pre-s monitor the PCR amplification,hybridization reactions and so on3.Microarray hybridization and detectionAfter DNA samples were obtained and amplified with PCR,the HBV PCR products were hybridized with oligonucleotide probes.The hybridized results were colorized by BCIP/NBT.According to the hybridization signal and the corresponding probe sequence,HBV genotype was determined.4.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.5.The repetition of genechips test.20 samples,which HBV-DNA was positive,were tested and 3 repeat tests were done for each samples and the results were analyzed.Results1.PCR primer and chip probe designWe have found 17 HBV genotypes genome in Genbank,including7 genotypes:A,B,C,D,E,F and G.Phylogenetic tree and the evolutionary relationships were analyzed.PCR primers and 7 probes were designed according to the difference of the sequences and the conservative consequences at HBV pre-S gene region.The product of PCR was 340bp.Blasting the probes in NCBI’Genbank and analyzing the first 50 sequences of the results,we found the accuracy of completely matching with our designed probes is 97%.2.Genotyping of clinical samplesWe tested the clinical DNA samples,which genotypes were determined with genome sequence comparison method.The microarray hybridization assay displayed a complete match to the previously identified genotypes;106 clinical serum samples collected from HBV infection individuals in Hangzhou.Among them,37and69 samples were positive to genotypes B and C.3.Validation of microarray genotypingThe results of hybrizations were identical with those of sequencing analysis.4.The repetition and sensitiveness of genechips test.20 samples,which HBV-DNA was positive,were tested and 3 repeat tests were done for each samples.The results are all positive.Of the 106 samples,,which HBV-DNA was positive,the lowest HBV-DNA title was 2~*10~4copies/ml,so we beliver the sensitiveness of the chips was not under the lever.Conclusion1.We have established a method of oligonucliotide probe designation according to the analysis of bioinformatics of HBV genome.2.We have fabricated microarray and begun to apply the chips for clinical use.It is demonstrated that this assay is a promising method for virus genotyping.3.HBV genotype B and C exsited in Hangzhou,and genotype C is a predominated genotype.However,To determine the distribution of HBV genotypes,we should take further investigation for HBV genotypes by enlarging study population.4.The assay is a simple to perform and can be used as a rapid,accurate and high-throughput method.It has a high clinical value in detecting the HBV genotype.更多还原