基于内部竞争性片段提高基因痕量突变检测方法的构建及临床运用

发布时间：2018-05-21 21:36

本文选题：KRAS基因 + BRAF基因　；参考：《第三军医大学》2016年博士论文

【摘要】：肿瘤是机体细胞因失去正常调控,因过度增殖而形成的新组织,且可侵犯周围组织或发生转移,肿瘤威胁着大众健康并给社会家庭带来沉重的负担。有数据显示,我国2015年因肿瘤死亡人数为281万,即平均每天死亡人数约7500人,肿瘤防控形势在我国依然严峻。尽早控制肿瘤发生、发展,降低其患病率与死亡率已经成为相关研究者急需解决的重大问题。随着分子生物学技术的发展,肿瘤相关基因位点的改变与肿瘤的发生、发展、诊治及其预后与有着密切的关系,其中肿瘤相关基因位点的改变即基因突变是指基因发生了碱基对组成或排列顺序的改变。能够引起肿瘤恶性增生的基因中,KRAS与其下游的核心分子BRAF基因突变是近年来研究的热点,它们任何一个的突变将促使丝裂原活化蛋白激酶途径持续性激活,导致组织朝恶性增生的方向发展。更有临床研究已经证明,由于KRAS基因的突变往往导致临床常用单克隆抗体药物治疗肿瘤的失效,美国食品药品监督管理局已经将其推荐为单抗药物用药前检查项目之一。随着临床标本的多样化及循环肿瘤DNA的深入研究,血液、组织及其它临床标本,如尿液、粪便、唾液、口腔粘膜脱落细胞等标本均可用于肿瘤源性突变DNA无创检测,早期基因突变的筛查能够为肿瘤早期诊断及预后提供重要依据。由于肿瘤相关的突变型基因,常隐藏于大量野生型基因中,如何在临床标本中有效检测出痕量的肿瘤源性的基因突变就十分重要。基因痕量突变的检测就能在大量的野生型基因的背景中,有效富集并找到发生痕量突变的目的基因,结合标本的检测数据,精确计算出其突变率,为肿瘤个体化治疗提供有力依据。在种类繁多的基因突变检测技术中,锁式PCR是检测基因痕量突变有效的方法之一,其主要原理是使用锁核酸探针提高碱基错配识别能力,使检测的灵敏度更高,符合基因突变检测的要求。然而,这种方法仍然存在诸多问题,如需要特异性的DNA聚合酶、对扩增的DNA质量要求高、扩增过程中有碱基人工错配导致目的基因非特异性扩增的现象等,鉴于此,本课题拟采用内部竞争性扩增片段的参与来降低检测过程中的碱基人工错配,避免目的基因非特异性扩增的现象,同时提高锁核酸探针对大量的野生型基因的屏蔽,凸显出基因痕量突变的突变位点,从而构建一种基于内部竞争性扩增片段提高野生型抑制性PCR(wirePCR),对基因痕量突变特异性扩增,通过实时荧光定量PCR达到基因痕量突变的检出。目的:1.根据肿瘤中常见的基因突变位点,结合内部竞争性扩增片段提高锁核酸抑制野生型背景基因的办法,构建结直肠肿瘤中KRAS与BRAF基因痕量突变的wirePCR高灵敏度检测方法,并对该方法反应条件进行优化。2.将构建的wire PCR检测方法进行系统评价,并用优化好的反应条件对临床肠镜活检标本中KRAS与BRAF基因痕量突变进行检测并做验证分析。3.进一步将内部竞争性扩增片段提高野生型抑制性方法运用于微滴数字PCR,用以检测循环肿瘤DNA基因痕量突变。4.构建内部竞争性扩增片段参与的野生型抑制性多重荧光PCR反应体系,达到多种基因痕量突变同时检测的目的。方法:1.设计合成引物对及荧光探针,对引物对和荧光探针浓度进行优化、采用温度梯度PCR对退火温度等条件进行优化;用已知KRAS和BRAF基因常见突变位点设计合成LNA/DNA嵌合体探针,以LEPTIN基因作为加入的内部竞争性扩增片段参与反应,构建内部竞争性扩增片段提高野生型抑制性基因痕量突变荧光定量检测体系。并进一步对反应体系的引物、荧光探针、锁核酸探针等反应条件进行优化。2.以突变型的的HT-29人结肠癌细胞系和野生型DNA按比例混合作为扩增模板,用上述构建好的方法及优化好的反应体系分别对突变率50%、25%、10%、1%、0.1%、0.01%的模板进行wire PCR反应,并将扩增产物进行并将扩增产物进行测序,评价内部竞争性扩增片段参与的野生型抑制性基因痕量突变检测方法的灵敏度、特异性、及准确性等指标。3.收集肠镜活检组织标本50例,检测DNA浓度并将浓度统一调整至100ng/ul,按照优化的wirePCR反应条件分别对临床标本进行KRAS和BRAF基因突变检测,同时结合20倍镜下切片HE染色病理分析结果和直接测序结果,对该方法检测结果进行验证。4.为了适应微滴数字PCR的反应条件和操作流程,试验中对KRAS、BRAF基因的上下游引物进行升级,选用具有更高TM值适应数字PCR的反应体系。相应地荧光探针也做了相应改进,选用带VIC和FAM荧光基团的MGB探针参与反应体系,用构建的内部竞争性扩增片段参与的野生型抑制性数字PCR检测标本中循环肿瘤DNA的KRAS、BRAF基因痕量突变情况。5.在内部竞争性扩增片段参与的野生型抑制性基因痕量突变检测的三重荧光反应体系中,内参LEPTIN基因作为加入的内部竞争性扩增片段,其引物及探针工作浓度与前期实验一致,KRAS、BRAF基因痕量突变的荧光探针中的荧光基团分别采用VIC、HEX、FAM,并两两交叉搭配,结合前期优化好的的引物和屏蔽野生型的锁核酸探针浓度参与反应。结果:1.完成了相关引物对、荧光探针、及锁核酸探针(LNA/DNA嵌合体)的设计与合成。加入反应体系的内部竞争性扩增片段引物对为HQ-329/330终浓度500nM荧光探针终浓度为100nM。确定了检测KRAS基因突变检测的最佳反应体系:引物对终浓度为500n M荧光探针终浓度250nM,针对KRAS基因野生型设计嵌合体探针HQ-144,其终浓度为500nM时能够对50-150 ng/μL的野生型模板能有效屏蔽;检测BRAF基因突变的最佳反应体系:引物对终浓度为500n M荧光探针终浓度250nM,针对其野生型设计嵌合体探针HQ-356,其终浓度为500nM时能够对50-200 ng/μL的野生型模板能有效屏蔽,在加入反应体系的内部竞争性扩增片段和屏蔽野生型锁核酸探针作用下,循环数60 cycles不会出现非特异性扩增的现象。2.在评价内部竞争性扩增片段参与的野生型抑制性基因痕量突变检测方法的灵敏度、特异性试验中,运用优化好的wirePCR反应条件,能够对突变比例只有0.01%的DNA模板有效检出,并有较好重复性,该方法标准曲线相关系数R2为0.996,拟合满意,扩增效率较高。3.用构建好的wirePCR方法对50例疑似结直肠癌肠镜组织进行检测,共检测出18例KRAS基因突变型(36%)和8例BRAF基因突变的标本(16%),本次检查突变标本均为KRAS或BRAF基因单一突变型,没有同时突变的现象,检测结果和切片HE染色镜下病理分析和直接测序结果相吻合。4.内部竞争性扩增片段参与的野生型抑制性微滴数字PCR中,体系中的引物终浓度均采用900n M,探针终浓度均采用200n M,模板DNA浓度为50 ng/μL。在检测KRAS基因痕量突变反应体系中,内部竞争性片段扩增引物对采用HQ-329/330,FAM荧光探针选用HQ-1433,KRAS基因引物对采用HQ-1595/1596,其VIC荧光探针选用HQ-1438,生成总油滴数1145800个,KRAS突变基因检出效率可以达到0.15%。在检测BRAF基因痕量突变反应体系中,内部竞争性片段荧光探针选用带有VIC基团的HQ-1434,BRAF基因引物对采用HQ-1592/1594,荧光探针选用带有FAM荧光基团HQ-671。生成总油滴数928355个,BRAF突变基因检出效率为0.11%。5.内部竞争性扩增片段参与的野生型抑制性基因痕量突变的三重荧光反应体系中,加入反应体系的内参LEPTIN基因和待测KRAS、BRAF基因引物对与前期实验条件一致,为了在相同CT值有相近的荧光强度,三重荧光反应体系中优化好的探针分别为:内部竞争性扩增片段即内参LEPTIN基因探针带有Texas Red荧光基团的探针HQ-1294探针终浓度为100n M,KRAS基因扩增检测中采用带有VIC荧光基团的MGB探针HQ-1438,BRAF基因采用带有FAM荧光基团的MGB探针HQ-671,荧光探针终浓度均为250n M,为避免反应中的非特异性扩增,退火温度选用60°C,循环数60 cycles。结论:1.构建基于内部竞争性扩增片段提高野生型抑制性特异性扩增基因痕量突变实时荧光定量检测方法,选出适合结直肠肿瘤中KRAS基因痕量突变检测的引物及探针,并对反应体系的退火温度,循环数等反应条件进行摸索,分别明确了加入内部竞争性扩增片段、KRAS基因检测的引物及探针最佳反应浓度。2.构建内部竞争性扩增片段提高野生型抑制性的BRAF基因基因痕量突变实时荧光定量检测方法,对引物、荧光探针和锁核酸探针进行一系列优化,使该反应体系可以对标本中的野生型DNA模板进行有效屏蔽,有较强选择性扩增能力,对BRAF基因突变识别灵敏度可以达到0.01%,满足基因痕量突变检测条件。3.将内部竞争性扩增片段提高野生型抑制性的基因痕量突变检测方法运用于50例临床结直肠肿瘤组织标本的KRAS和BRAF基因痕量突变检测,本批标本中突变率分别为36%和16%,该方法灵敏度特异性高、操作与成本比直接测序法更有优势,可广泛应用于临床基因突变检测,为肿瘤监测及个体化用药提供参考。4.内部竞争性扩增片段提高野生型抑制性的基因痕量突变检测方法结合微滴数字PCR可以检测循环肿瘤DNA的痕量突变,通过对野生型基因扩增的抑制,可以达到单拷贝数基因突变的绝对定量检测,也可用来评估相关基因痕量突变检测方法的灵敏度。5.内部竞争性扩增片段提高野生型抑制性的三重荧光基因痕量突变检测结果提示,构建的基因痕量突变检测方法可以运用于多种基因痕量突变的联合检测,试验中闭管操作,在相同反应条件下的检测的数据结果更精确,且大大缩短反应时间,减少实验成本和重复性实验操作,有较强的临床使用价值。
[Abstract]:Tumor is a new tissue caused by the loss of normal control and excessive proliferation. It can invade the surrounding tissue or transfer. The tumor threatens the public health and brings heavy burden to the social family. The number of cancer deaths in 2015 in China is 2 million 810 thousand, that is, the average number of deaths per day of about 7500 people, tumor prevention and control. The situation is still grim in China. Early control of tumor occurrence, development, reduction of its morbidity and mortality has become a major problem that the related researchers need to solve. With the development of molecular biology technology, the change of tumor related gene loci is closely related to the occurrence, development, diagnosis and prognosis of tumor. The change in the gene loci, that is, the mutation of the gene is the change in the composition or order of the base pairs. In the genes that can cause malignant proliferation of the tumor, the mutation of KRAS and its downstream core molecule BRAF gene is a hot spot in recent years. Any mutation of these genes will promote the persistent excitation of the mitogen activated protein kinase pathway. More clinical studies have proved that the KRAS gene mutation often leads to the failure of the clinical commonly used monoclonal antibody drugs for the treatment of tumors. The US Food and drug administration has recommended it as one of the pre drug test items for McAbs. With the diversification of clinical specimens, The in-depth study of circulating tumor DNA, blood, tissue and other clinical specimens, such as urine, feces, saliva, oral mucosa exfoliative cells and other specimens can be used for noninvasive detection of tumor derived mutation DNA. Early gene mutation screening can provide a heavy basis for early diagnosis and prognosis of tumor. In a large number of wild genotypes, it is very important to detect trace mutations in a tumor derived gene effectively in a clinical specimen. The detection of trace mutations in the gene can effectively enrich and find a target gene for trace mutations in the background of a large number of wild type genes. In a wide variety of gene mutation detection techniques, locked PCR is one of the effective methods for detecting trace mutations in genes. The main principle is to use a nucleic acid probe to improve the ability of base mismatch recognition, to make the detection more sensitive and meet the requirements of gene mutation detection. There are still many problems in the method, such as the need for specific DNA polymerase, high requirement for the mass of DNA and the phenomenon of non specific amplification of the target gene in the process of amplification in the process of amplification. In view of this, this subject is intended to use the participation of competitive amplified fragment to reduce the base artificial mismatch in the detection process and avoid the target. The phenomenon of non specific amplification of genes, at the same time, enhances the shielding of a large number of wild type genes by the nucleic acid probe and highlights the mutation site of the trace mutation, thus building a kind of wild type inhibitory PCR (wirePCR) based on the internal competitive amplification fragment, the specific amplification of the trace mutation of the gene, and the real-time quantitative PCR by quantitative fluorescence. Detection of trace mutations in the gene. Objective: 1. a high sensitivity method for detecting trace mutations in KRAS and BRAF genes in colorectal tumors was constructed according to the common gene mutation sites in the tumor, combined with the internal competitive amplification fragment to improve the inhibition of the wild type background gene of the nucleic acid in the colorectal tumor, and the reaction conditions were optimized by.2.. The wire PCR detection method is systematically evaluated and the optimized reaction conditions are used to detect and verify the trace mutations of the KRAS and BRAF genes in the clinical enteroscopy specimens..3. is used to further improve the internal competitive amplification fragment to improve the wild type inhibition method to the micro drop digital PCR, so as to detect the DNA base of the circulating tumor. The aim of the simultaneous detection of trace mutations in a variety of genes is achieved by constructing an internal competitive multiplex multiple fluorescence PCR reaction system in which an internal competitive amplification fragment is constructed with trace mutation.4.. Method: 1. the primer pairs and fluorescent probes are designed and synthesized. The concentration of primer pairs and fluorescent probes is optimized, and the temperature gradient PCR is used for annealing temperature and other conditions. In line optimization, the LNA/DNA chimerism probe was designed and synthesized with the common mutation sites of the known KRAS and BRAF genes, and the LEPTIN gene was used as an internal competitive amplified fragment to participate in the reaction. The internal competitive amplification fragment was constructed to improve the fluorescence quantitative detection system of the wild type inhibitory gene, and the primers and fluores of the reaction system were further amplified. The reaction conditions such as light probe, nucleic acid probe and other reaction conditions were optimized by.2., the mutant HT-29 human colon cancer cell line and wild type DNA were mixed as the amplification template. The wire PCR reaction was carried out on the template of the mutation rate 50%, 25%, 10%, 1%, 0.1%, 0.01%, respectively. The amplification products were sequenced and the sensitivity, specificity, and accuracy of the detection methods of wild type inhibitory gene mutations involved in the internal competitive amplified fragment were collected in 50 cases of enteroscopy biopsy tissue specimens, and the concentration of DNA was detected and the concentration was adjusted to 100ng/ul, and the optimal wirePCR reaction conditions were applied to the.3.. KRAS and BRAF gene mutations were detected in clinical specimens, combined with pathological analysis results of HE staining under 20 times microscope and direct sequencing results, the results were verified by.4. in order to adapt to the reaction conditions and operation process of microdrop digital PCR. In the experiment, the upstream and downstream primers of KRAS, BRAF base were upgraded, and higher T was selected. The M value adapts to the response system of the digital PCR. The corresponding fluorescence probe has also made a corresponding improvement. The MGB probe with VIC and FAM fluorescent groups is selected to participate in the reaction system. The KRAS of the circulating tumor DNA in the specimens is detected by the constructed endogenous competitive amplification fragment involved in the wild type inhibitory digital PCR, and the BRAF gene trace mutation.5. is competitive in the internal competition. In the three heavy fluorescence reaction system for the detection of trace mutation of wild type inhibitory gene, the LEPTIN gene was used as an internal competitive amplification fragment, and the working concentration of primers and probes was consistent with the previous experiment. The fluorescence groups of the trace mutations of KRAS and BRAF genes were VIC, HEX, FAM, respectively. 22 cross matching, combined with pre optimized primers and shielding wild type nucleic acid probe concentration to participate in the reaction. Results: 1. the design and synthesis of related primer pairs, fluorescent probes, and nucleic acid probe (LNA/DNA chimerism) were completed. The internal competitive amplified fragment primers for the reaction system were 500nM fluorescence at the final concentration of HQ-329/330. The final concentration of the probe was 100nM. to determine the best reaction system for detecting KRAS gene mutation detection: the final concentration of the primer pair was 500N M fluorescence probe final concentration 250nM, the KRAS gene wild-type chimerism probe HQ-144 was designed, and the final concentration was 500nM capable of shielding the wild type template of 50-150 ng/ L, and detecting the most significant mutation of the BRAF gene. The best reaction system: the final concentration of the primer pair is 500N M fluorescence probe final concentration 250nM, which is designed for its wild type chimerism probe HQ-356. When the final concentration is 500nM, the wild type template of 50-200 ng/ mu L can be effectively shielded. The cycle number is 6 under the action of the internal competitive amplification fragment and the shielding wild type nucleic acid probe. 0 cycles does not appear nonspecific amplification..2. is sensitive to the detection of trace mutation detection methods of wild type inhibitory genes involved in the internal competitive amplification fragments. In the specificity test, the optimized wirePCR reaction conditions can be used to detect the DNA template with a mutation ratio of only 0.01%, and have good reproducibility. The standard curve correlation coefficient R2 of the method was 0.996, the fitting was satisfactory and the amplification efficiency was higher.3. using the constructed wirePCR method to detect 50 cases of suspected colorectal carcinoma enteroscopy, 18 cases of KRAS mutation type (36%) and 8 cases of BRAF gene mutation were detected (16%). All of these specimens were single mutant of KRAS or BRAF gene. At the same time, the phenomenon of mutation, the detection results, the pathological analysis of the HE staining microscope and the direct sequencing results coincide with the wild inhibitory microdrop digital PCR involved in the competitive amplified fragment of.4.. The final concentration of primers in the system uses 900N M, the final concentration of the probe uses 200N M, and the concentration of the template DNA is 50 ng/ L. in the detection of the KRAS gene mark. In the system of mutational response, the internal competitive fragment amplification primers used HQ-329/330, FAM fluorescence probe to select HQ-1433, KRAS gene primers for HQ-1595/1596, and HQ-1438 for the VIC fluorescence probe. The number of total oil droplets was 1145800, and the detection efficiency of KRAS mutation gene could reach 0.15%. in the detection of trace mutation reaction system of BRAF gene. The internal competitive fragment fluorescent probe selects HQ-1434 with VIC group, BRAF gene primers to HQ-1592/1594, fluorescent probe with FAM fluorescent group HQ-671. to generate 928355 total oil droplets, and the detection efficiency of BRAF mutation gene is three of the trace mutation of wild type inhibitory gene involved in the internal competitive amplification fragment of 0.11%.5.. In the fluorescent reaction system, the internal reference LEPTIN gene of the reaction system and the KRAS, BRAF primers are consistent with the previous experimental conditions. In order to have similar fluorescence intensity at the same CT value, the optimized probe in the three heavy fluorescence reaction system is the internal competitive amplification fragment, the internal reference LEPTIN gene probe with the Texas Red fluorescence. The final concentration of the probe HQ-1294 probe of the group is 100N M, and the MGB probe HQ-1438 with VIC fluorescent group is used in the KRAS gene amplification detection, BRAF gene adopts MGB probe with FAM fluorescent group, and the final concentration of the fluorescent probe is 250N, in order to avoid the non-specific expansion in the reaction, the annealing temperature is selected 60 degrees, and the cycle number is 60 conclusions: 1. a real-time quantitative detection method based on an internal competitive amplification fragment to improve the trace mutation of the wild type inhibitory specific amplification gene was established, and the primers and probes were selected for the detection of trace mutations of the KRAS gene in the colorectal tumor, and the reaction conditions of the annealing temperature and the cycle number of the reaction system were explored, respectively. The internal competitive amplification fragments, the primers detected by KRAS gene and the best reaction concentration of the probe.2. construct the internal competitive amplification fragment to improve the real-time fluorescence quantitative detection method of the wild type BRAF gene trace mutation, and make a series of optimization on the primers, fluorescent probes and nucleic acid probes, so that the reaction system can be used to the specimen. The wild type DNA template is effectively screened, has a strong selective amplification ability, and the sensitivity of BRAF gene mutation identification can reach 0.01%. It satisfies the trace mutation detection condition of the gene.3. and uses the internal competitive amplification fragment to improve the wild type of the gene trace mutation detection method for the 50 cases of clinical colorectal tumor tissue specimens. The detection of trace mutations of KRAS and BRAF genes in this group is 36% and 16%, respectively. The sensitivity of this method is 36% and 16%, and the sensitivity of the method is high. The operation and cost are more advantageous than the direct sequencing. It can be widely used in the detection of clinical gene mutation, and it provides the internal competitive amplification fragment of.4. to improve the wild type inhibition for tumor monitoring and individualized drug use. The trace mutation detection method combined with the microsatellite PCR can detect the trace mutations of the circulating tumor DNA. By inhibiting the amplification of the wild type gene, it can reach the absolute quantitative detection of the mutation of the single copy number gene, and can also be used to evaluate the sensitivity of the sensitivity.5. in the detection of the trace mutation of the related genes. The results of trace mutation detection of high wild type three heavy fluorescence gene suggest that the method of detection of trace mutation can be used for joint detection of trace mutations in many genes. In the test, the result of closed tube operation, the result of detection under the same reaction condition is more accurate, and the reaction time is greatly shortened, and the cost and weight of the experiment are reduced. The complex experimental operation has a strong clinical value.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R73-3

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