【作者】 李娟；

【导师】 杜红丽； Chen Allen Menglin；

【作者基本信息】 华南理工大学 ， 工程硕士（专业学位）， 2018， 硕士

【摘要】 近年来,随着精准医疗的兴起,各种分子诊断技术迅速发展。以基因测序为代表的医疗技术受到各方的关注,开始迎来全面的爆发式增长。Sanger测序技术自出现以来,一直以准确性作为分子诊断行业的金标准。单核苷酸多态性即SNP在病毒耐药及分型、疾病易感基因、药物敏感性、肿瘤靶向用药、人类进化和群体遗传学研究中起重要作用。随着病毒微生物的进化,环境和饮食等因素造成人类基因结构的改变引起Sanger测序数据的数量、难度和复杂度的不断增加,更重要的是,测序灵敏度一直被诟病,科研临床工作者必须借助生物信息学工具对数据进行分析和处理,才能得到临床有用信息。这些工具虽然对用户隐藏了复杂的计算过程,但是在操作使用方面存在局限,生物数据的管理与处理通常是在linux环境下进行的,其专业化的操作界面亦不为临床工作者所熟知。传染病是威胁人类健康的一类重大疾病,快速、准确的诊断是有效治疗、病情监测和控制疾病蔓延的重要前提。随着分子检测技术的不断发展和完善,分子检测在病原微生物感染诊断及治疗监测的临床应用日益广泛,已成为传染病诊断和疗效评价中的重要工具。2017年12月1日,国家卫计委发布的感染性疾病相关个体化医学分子检测技术指南指出DNA测序技术是现阶段感染性疾病检测的金标准,但临床治疗中遇到低丰度的基因突变却常常不能被识别。比如慢性乙型肝炎早期耐药研究的初治患者外周血样本,常常包含了早期病毒,疑似病毒成分病人的健康外周血。为此,一个操作简单,界面友好且功能强大的分析工具,能够更好的解决早期诊断中灵敏度问题。本文的主要内容可以分为三个部分,第一部分简单介绍了Sanger测序方法灵敏度研究的背景,传染病临床耐药目前面临的挑战,Sanger数据分析的现状及目前常用峰图数据软件在低频测序峰图的处理上存在的局限性和不足,提出ab1 peak reporter分析工具低丰度突变定量研究在传染病临床耐药治疗中必要性和意义。第二部分以人P53基因突变体作为标准品测试分析工具的可行性,介绍Thermo Fisher Scientific网站的在线分析工具ab1 peak reporter,以已知突变灵敏度的人P53基因测试样本测序数据作为内参,构建8种不同比例的野生株和突变株,两组平行实验用ab1peak reporter分析工具和seqscanner工具输出各自的电泳图,实现突变丰度的定量,通过查找保守基因序列,实现突变位点分析,通过相关函数系数验证ab1 peak reporter工具在不同混合比例情况下与已知突变体的相关性和准确度。本文第三部分以传染病临床最具代表性的慢性乙型肝炎耐药和结核分枝杆菌利福平临床耐药疾病作为研究对象,各选取一定的临床数据进行实验验证,结果表明ab1 peak reporter能很好地辅助临床进行两种疾病耐药的早期诊断和低丰度的研究,为临床学者提供方便,是Sanger测序数据处理和分析的有效工具,主要内容和方法为:选取符合Sanger测序方法最低检测限核酸定量为1*10~3的深圳市人民医院慢性乙型肝炎耐药临床血液标本27例,采用离心柱法提取乙肝DNA核酸,随后参照中山大学达安基因生产的乙肝病毒耐药基因突变检测试剂盒(PCR-测序法)的说明书对乙肝病毒基因P区进行PCR扩增,凝胶糖电泳和纯化,上机毛细管电泳3500DX基因分析仪,27例测序结果ab1数据上传ab1 peak reporter进行分析,结果与基因分析仪配套的软件Seqscanner对比,ab1 peak reporter工具可以实现突变丰度的定量,帮助临床发现了11例低频突变,提高了检出率,减少了差错的发生概率,其中5名慢性乙型肝炎患者因提早获知低丰度药物耐药突变,更换药物治疗方案,使患者和临床受益。选取符合Sanger测序方法最低检测限核酸定量为1*10~3的广州医科大学附属第一医院结核肺结核分枝杆菌利福平耐药临床案例24例,从痰液提取结核分枝杆菌DNA,随后参照中山大学达安基因的结核分枝杆菌利福平耐药突变检测试剂盒(PCR-Sanger测序法)的说明书对结核分枝杆菌利福平耐药关联基因rpoB基因进行扩增,电泳和纯化,上机3500DX基因分析仪,24例测序结果ab1数据上传ab1 peak reporter进行分析,结果与基因分析仪配套的软件Seqscanner对比,ab1 peak reporter工具帮助临床发现9例低丰度耐药突变,其中2名患者可根据低丰度耐药突变提早更换药物治疗方案,提高治疗效果节省治疗费用,有良好的应用价值。更多还原

【Abstract】 In recent years,with the rise of precision medicine,a variety of molecular diagnostic technology has developed rapidly.The medical technology,represented by gene sequencing,has been concerned by all parties,starting to usher in the day of a comprehensively explosive growth.Since Sanger sequencing technology emerges in early period,accuracy has been regarded as the gold standard of molecular diagnosis industry.Single nucleotide polymorphism(SNP),that is,SNP plays an important role in drug resistance and typing of viruses,susceptibility genes,drug sensitivity,tumor targeting drugs,human evolution and population genetics.With the evolution of virus microbes,changes in the structure of human genes caused by environmental and dietary factors have increased the amount,difficulty and complexity of Sanger sequencing data.More importantly,the sensitivity of Sanger sequencing has been criticized all the time.In order to obtain useful clinical information,researchers must use bioinformatics tools to analyze and process the data.Although these tools hide complex computing process from users,but there are limitations in the operational use,the management and processing of biological data is usually carried out in the linux environment,and its specialized operation interface is not well known to clinicians.Infectious disease is a major disease threatening human health,rapid and accurate diagnosis is an important prerequisite to effective treatment,disease monitoring and control of the spread of disease.With the development and ever-increasing improvement of molecular detection technology,molecular detection has become an important tool in the diagnosis of infectious diseases and the evaluation of curative effect.In December 1st 2017,the National Health and Family Planning Commission issued the guidelines for individualized Medical Molecular Detection of Infectious Diseases,pointing out that DNA sequencing technology is the gold standard for infectious disease detection at this stage.However,gene mutations with low abundance are often not recognized in clinical therapy.For example,the peripheral blood samples of patients with early drug resistance studies of chronic hepatitis B often contain the healthy peripheral blood of patients with early stage viruses,suspected viral components.Therefore,a simple,user-friendly and powerful analysis tool can better solve the sensitivity problem in early diagnosis.The main contents of this paper can be divided into three parts.The first part briefly introduces the background of sensitivity research of Sanger sequencing method.The current challenges of clinical drug resistance in infectious diseases are the current situation of Sanger data analysis and the limitations and shortcomings of current software in processing low-frequency sequencing peak.The necessity and significance of ab1 peak reporter analysis tool for quantitative analysis of low level mutation in clinical drug resistance treatment of infectious diseases were put forward.In the second part,the feasibility of using human p53 gene mutants as standard test tool is introduced.Ab1 peak reporter,an online analysis tool of Thermo Fisher Scientific website,and sequencing data of human p53 gene test samples with known mutation sensitivity are used as internal reference.Eight different proportions of wild and mutant strains were constructed.Two groups of parallel experiments used ab1 peak reporter analysis and seqscanner to output their respective electrophoretic patterns to realize the quantitative analysis of mutation,and to analyze the mutation sites by searching for conservative gene sequences.Correlation coefficient was used to verify the correlation and accuracy of ab1 peak reporter with known mutants in different mixing proportions.In the third part of this paper,the most representative chronic hepatitis B drug resistant disease and rifampicin resistant disease of Mycobacterium tuberculosis were selected as the research objects,and some clinical data were selected for experimental verification.The results show that ab1 peak reporter can assist in the early diagnosis and low level mutation study of two kinds of disease resistance,and is an effective tool for Sanger sequencing data processing and analysis.The main contents and methods are as follows:A total of 27 clinical blood samples of chronic hepatitis B resistance in Shenzhen Hospital were selected to extract hepatitis B DNA nucleic acid(namely 1*10~3)by centrifugal-column method,which accord with the lowest detection limit nucleic acid of Sanger sequencing method.Then referring to the mutation detection kit of hepatitis B virus resistance gene produced by Daan Gene of Sun Yat-sen University(PCR-sequencing)PCR amplification,gel electrophoresis and purification of P region of hepatitis B virus gene together with capillary electrophoresis of 3500DX gene analyzer,those were used to analyze27 cases of ab1 data uploaded to ab1 peak reporter.Results compared with the Seqscanner of gene analyzer,the ab1 peak reporter tool can realize the quantitative analysis of mutation abundance,help to find 11 cases of low level mutation,improve the detection rate and reduce the probability of error.Among them,5 patients with chronic hepatitis B received low level drug resistance mutations in advance,and changed drug treatment protocols for the benefit of patients and clinical practice.24 cases of rifampicin resistance of Mycobacterium tuberculosis in the first affiliated Hospital of Guangzhou Medical University were selected.The DNA of Mycobacterium tuberculosis was extracted from sputum,then the rifampicin mutation test kit of Sun Yat-sen University Daan gene(PCR-Sanger sequencing)was used to detect the rifampicin resistance of Mycobacterium tuberculosis and amplify The rpoB gene of rifampin resistance associated gene of Mycobacterium tuberculosis with electrophoretic and purified practice.The ab1 data of 24 patients with rifampicin resistance associated gene were uploaded to ab1 peak reporter and analyzed by using 3500DX gene analyzer.Results compared with the software of gene analyzer,Seqscanner,the ab1 peak reporter tool was used to find 9 cases of low abundance resistance mutation in clinic.Two of the patients could change the drug treatment plan early according to the low abundance resistance mutation,so as to improve the therapeutic effect and save the treatment cost.It has good application value.更多还原