本文選題：KRAS基因 + BRAF基因　； 參考：《第三軍醫(yī)大學(xué)》2016年博士論文

【摘要】：腫瘤是機(jī)體細(xì)胞因失去正常調(diào)控,因過(guò)度增殖而形成的新組織,且可侵犯周圍組織或發(fā)生轉(zhuǎn)移,腫瘤威脅著大眾健康并給社會(huì)家庭帶來(lái)沉重的負(fù)擔(dān)。有數(shù)據(jù)顯示,我國(guó)2015年因腫瘤死亡人數(shù)為281萬(wàn),即平均每天死亡人數(shù)約7500人,腫瘤防控形勢(shì)在我國(guó)依然嚴(yán)峻。盡早控制腫瘤發(fā)生、發(fā)展,降低其患病率與死亡率已經(jīng)成為相關(guān)研究者急需解決的重大問(wèn)題。隨著分子生物學(xué)技術(shù)的發(fā)展,腫瘤相關(guān)基因位點(diǎn)的改變與腫瘤的發(fā)生、發(fā)展、診治及其預(yù)后與有著密切的關(guān)系,其中腫瘤相關(guān)基因位點(diǎn)的改變即基因突變是指基因發(fā)生了堿基對(duì)組成或排列順序的改變。能夠引起腫瘤惡性增生的基因中,KRAS與其下游的核心分子BRAF基因突變是近年來(lái)研究的熱點(diǎn),它們?nèi)魏我粋�(gè)的突變將促使絲裂原活化蛋白激酶途徑持續(xù)性激活,導(dǎo)致組織朝惡性增生的方向發(fā)展。更有臨床研究已經(jīng)證明,由于KRAS基因的突變往往導(dǎo)致臨床常用單克隆抗體藥物治療腫瘤的失效,美國(guó)食品藥品監(jiān)督管理局已經(jīng)將其推薦為單抗藥物用藥前檢查項(xiàng)目之一。隨著臨床標(biāo)本的多樣化及循環(huán)腫瘤DNA的深入研究,血液、組織及其它臨床標(biāo)本,如尿液、糞便、唾液、口腔粘膜脫落細(xì)胞等標(biāo)本均可用于腫瘤源性突變DNA無(wú)創(chuàng)檢測(cè),早期基因突變的篩查能夠?yàn)槟[瘤早期診斷及預(yù)后提供重要依據(jù)。由于腫瘤相關(guān)的突變型基因,常隱藏于大量野生型基因中,如何在臨床標(biāo)本中有效檢測(cè)出痕量的腫瘤源性的基因突變就十分重要。基因痕量突變的檢測(cè)就能在大量的野生型基因的背景中,有效富集并找到發(fā)生痕量突變的目的基因,結(jié)合標(biāo)本的檢測(cè)數(shù)據(jù),精確計(jì)算出其突變率,為腫瘤個(gè)體化治療提供有力依據(jù)。在種類繁多的基因突變檢測(cè)技術(shù)中,鎖式PCR是檢測(cè)基因痕量突變有效的方法之一,其主要原理是使用鎖核酸探針提高堿基錯(cuò)配識(shí)別能力,使檢測(cè)的靈敏度更高,符合基因突變檢測(cè)的要求。然而,這種方法仍然存在諸多問(wèn)題,如需要特異性的DNA聚合酶、對(duì)擴(kuò)增的DNA質(zhì)量要求高、擴(kuò)增過(guò)程中有堿基人工錯(cuò)配導(dǎo)致目的基因非特異性擴(kuò)增的現(xiàn)象等,鑒于此,本課題擬采用內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段的參與來(lái)降低檢測(cè)過(guò)程中的堿基人工錯(cuò)配,避免目的基因非特異性擴(kuò)增的現(xiàn)象,同時(shí)提高鎖核酸探針對(duì)大量的野生型基因的屏蔽,凸顯出基因痕量突變的突變位點(diǎn),從而構(gòu)建一種基于內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段提高野生型抑制性PCR(wirePCR),對(duì)基因痕量突變特異性擴(kuò)增,通過(guò)實(shí)時(shí)熒光定量PCR達(dá)到基因痕量突變的檢出。目的:1.根據(jù)腫瘤中常見(jiàn)的基因突變位點(diǎn),結(jié)合內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段提高鎖核酸抑制野生型背景基因的辦法,構(gòu)建結(jié)直腸腫瘤中KRAS與BRAF基因痕量突變的wirePCR高靈敏度檢測(cè)方法,并對(duì)該方法反應(yīng)條件進(jìn)行優(yōu)化。2.將構(gòu)建的wire PCR檢測(cè)方法進(jìn)行系統(tǒng)評(píng)價(jià),并用優(yōu)化好的反應(yīng)條件對(duì)臨床腸鏡活檢標(biāo)本中KRAS與BRAF基因痕量突變進(jìn)行檢測(cè)并做驗(yàn)證分析。3.進(jìn)一步將內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段提高野生型抑制性方法運(yùn)用于微滴數(shù)字PCR,用以檢測(cè)循環(huán)腫瘤DNA基因痕量突變。4.構(gòu)建內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段參與的野生型抑制性多重?zé)晒釶CR反應(yīng)體系,達(dá)到多種基因痕量突變同時(shí)檢測(cè)的目的。方法:1.設(shè)計(jì)合成引物對(duì)及熒光探針,對(duì)引物對(duì)和熒光探針濃度進(jìn)行優(yōu)化、采用溫度梯度PCR對(duì)退火溫度等條件進(jìn)行優(yōu)化;用已知KRAS和BRAF基因常見(jiàn)突變位點(diǎn)設(shè)計(jì)合成LNA/DNA嵌合體探針,以LEPTIN基因作為加入的內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段參與反應(yīng),構(gòu)建內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段提高野生型抑制性基因痕量突變熒光定量檢測(cè)體系。并進(jìn)一步對(duì)反應(yīng)體系的引物、熒光探針、鎖核酸探針等反應(yīng)條件進(jìn)行優(yōu)化。2.以突變型的的HT-29人結(jié)腸癌細(xì)胞系和野生型DNA按比例混合作為擴(kuò)增模板,用上述構(gòu)建好的方法及優(yōu)化好的反應(yīng)體系分別對(duì)突變率50%、25%、10%、1%、0.1%、0.01%的模板進(jìn)行wire PCR反應(yīng),并將擴(kuò)增產(chǎn)物進(jìn)行并將擴(kuò)增產(chǎn)物進(jìn)行測(cè)序,評(píng)價(jià)內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段參與的野生型抑制性基因痕量突變檢測(cè)方法的靈敏度、特異性、及準(zhǔn)確性等指標(biāo)。3.收集腸鏡活檢組織標(biāo)本50例,檢測(cè)DNA濃度并將濃度統(tǒng)一調(diào)整至100ng/ul,按照優(yōu)化的wirePCR反應(yīng)條件分別對(duì)臨床標(biāo)本進(jìn)行KRAS和BRAF基因突變檢測(cè),同時(shí)結(jié)合20倍鏡下切片HE染色病理分析結(jié)果和直接測(cè)序結(jié)果,對(duì)該方法檢測(cè)結(jié)果進(jìn)行驗(yàn)證。4.為了適應(yīng)微滴數(shù)字PCR的反應(yīng)條件和操作流程,試驗(yàn)中對(duì)KRAS、BRAF基因的上下游引物進(jìn)行升級(jí),選用具有更高TM值適應(yīng)數(shù)字PCR的反應(yīng)體系。相應(yīng)地?zé)晒馓结樢沧隽讼鄳?yīng)改進(jìn),選用帶VIC和FAM熒光基團(tuán)的MGB探針參與反應(yīng)體系,用構(gòu)建的內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段參與的野生型抑制性數(shù)字PCR檢測(cè)標(biāo)本中循環(huán)腫瘤DNA的KRAS、BRAF基因痕量突變情況。5.在內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段參與的野生型抑制性基因痕量突變檢測(cè)的三重?zé)晒夥磻?yīng)體系中,內(nèi)參LEPTIN基因作為加入的內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段,其引物及探針工作濃度與前期實(shí)驗(yàn)一致,KRAS、BRAF基因痕量突變的熒光探針中的熒光基團(tuán)分別采用VIC、HEX、FAM,并兩兩交叉搭配,結(jié)合前期優(yōu)化好的的引物和屏蔽野生型的鎖核酸探針濃度參與反應(yīng)。結(jié)果:1.完成了相關(guān)引物對(duì)、熒光探針、及鎖核酸探針(LNA/DNA嵌合體)的設(shè)計(jì)與合成。加入反應(yīng)體系的內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段引物對(duì)為HQ-329/330終濃度500nM熒光探針終濃度為100nM。確定了檢測(cè)KRAS基因突變檢測(cè)的最佳反應(yīng)體系:引物對(duì)終濃度為500n M熒光探針終濃度250nM,針對(duì)KRAS基因野生型設(shè)計(jì)嵌合體探針HQ-144,其終濃度為500nM時(shí)能夠?qū)?0-150 ng/μL的野生型模板能有效屏蔽;檢測(cè)BRAF基因突變的最佳反應(yīng)體系:引物對(duì)終濃度為500n M熒光探針終濃度250nM,針對(duì)其野生型設(shè)計(jì)嵌合體探針HQ-356,其終濃度為500nM時(shí)能夠?qū)?0-200 ng/μL的野生型模板能有效屏蔽,在加入反應(yīng)體系的內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段和屏蔽野生型鎖核酸探針作用下,循環(huán)數(shù)60 cycles不會(huì)出現(xiàn)非特異性擴(kuò)增的現(xiàn)象。2.在評(píng)價(jià)內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段參與的野生型抑制性基因痕量突變檢測(cè)方法的靈敏度、特異性試驗(yàn)中,運(yùn)用優(yōu)化好的wirePCR反應(yīng)條件,能夠?qū)ν蛔儽壤挥?.01%的DNA模板有效檢出,并有較好重復(fù)性,該方法標(biāo)準(zhǔn)曲線相關(guān)系數(shù)R2為0.996,擬合滿意,擴(kuò)增效率較高。3.用構(gòu)建好的wirePCR方法對(duì)50例疑似結(jié)直腸癌腸鏡組織進(jìn)行檢測(cè),共檢測(cè)出18例KRAS基因突變型(36%)和8例BRAF基因突變的標(biāo)本(16%),本次檢查突變標(biāo)本均為KRAS或BRAF基因單一突變型,沒(méi)有同時(shí)突變的現(xiàn)象,檢測(cè)結(jié)果和切片HE染色鏡下病理分析和直接測(cè)序結(jié)果相吻合。4.內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段參與的野生型抑制性微滴數(shù)字PCR中,體系中的引物終濃度均采用900n M,探針終濃度均采用200n M,模板DNA濃度為50 ng/μL。在檢測(cè)KRAS基因痕量突變反應(yīng)體系中,內(nèi)部競(jìng)爭(zhēng)性片段擴(kuò)增引物對(duì)采用HQ-329/330,FAM熒光探針選用HQ-1433,KRAS基因引物對(duì)采用HQ-1595/1596,其VIC熒光探針選用HQ-1438,生成總油滴數(shù)1145800個(gè),KRAS突變基因檢出效率可以達(dá)到0.15%。在檢測(cè)BRAF基因痕量突變反應(yīng)體系中,內(nèi)部競(jìng)爭(zhēng)性片段熒光探針選用帶有VIC基團(tuán)的HQ-1434,BRAF基因引物對(duì)采用HQ-1592/1594,熒光探針選用帶有FAM熒光基團(tuán)HQ-671。生成總油滴數(shù)928355個(gè),BRAF突變基因檢出效率為0.11%。5.內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段參與的野生型抑制性基因痕量突變的三重?zé)晒夥磻?yīng)體系中,加入反應(yīng)體系的內(nèi)參LEPTIN基因和待測(cè)KRAS、BRAF基因引物對(duì)與前期實(shí)驗(yàn)條件一致,為了在相同CT值有相近的熒光強(qiáng)度,三重?zé)晒夥磻?yīng)體系中優(yōu)化好的探針?lè)謩e為:內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段即內(nèi)參LEPTIN基因探針帶有Texas Red熒光基團(tuán)的探針HQ-1294探針終濃度為100n M,KRAS基因擴(kuò)增檢測(cè)中采用帶有VIC熒光基團(tuán)的MGB探針HQ-1438,BRAF基因采用帶有FAM熒光基團(tuán)的MGB探針HQ-671,熒光探針終濃度均為250n M,為避免反應(yīng)中的非特異性擴(kuò)增,退火溫度選用60°C,循環(huán)數(shù)60 cycles。結(jié)論:1.構(gòu)建基于內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段提高野生型抑制性特異性擴(kuò)增基因痕量突變實(shí)時(shí)熒光定量檢測(cè)方法,選出適合結(jié)直腸腫瘤中KRAS基因痕量突變檢測(cè)的引物及探針,并對(duì)反應(yīng)體系的退火溫度,循環(huán)數(shù)等反應(yīng)條件進(jìn)行摸索,分別明確了加入內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段、KRAS基因檢測(cè)的引物及探針最佳反應(yīng)濃度。2.構(gòu)建內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段提高野生型抑制性的BRAF基因基因痕量突變實(shí)時(shí)熒光定量檢測(cè)方法,對(duì)引物、熒光探針和鎖核酸探針進(jìn)行一系列優(yōu)化,使該反應(yīng)體系可以對(duì)標(biāo)本中的野生型DNA模板進(jìn)行有效屏蔽,有較強(qiáng)選擇性擴(kuò)增能力,對(duì)BRAF基因突變識(shí)別靈敏度可以達(dá)到0.01%,滿足基因痕量突變檢測(cè)條件。3.將內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段提高野生型抑制性的基因痕量突變檢測(cè)方法運(yùn)用于50例臨床結(jié)直腸腫瘤組織標(biāo)本的KRAS和BRAF基因痕量突變檢測(cè),本批標(biāo)本中突變率分別為36%和16%,該方法靈敏度特異性高、操作與成本比直接測(cè)序法更有優(yōu)勢(shì),可廣泛應(yīng)用于臨床基因突變檢測(cè),為腫瘤監(jiān)測(cè)及個(gè)體化用藥提供參考。4.內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段提高野生型抑制性的基因痕量突變檢測(cè)方法結(jié)合微滴數(shù)字PCR可以檢測(cè)循環(huán)腫瘤DNA的痕量突變,通過(guò)對(duì)野生型基因擴(kuò)增的抑制,可以達(dá)到單拷貝數(shù)基因突變的絕對(duì)定量檢測(cè),也可用來(lái)評(píng)估相關(guān)基因痕量突變檢測(cè)方法的靈敏度。5.內(nèi)部競(jìng)爭(zhēng)性擴(kuò)增片段提高野生型抑制性的三重?zé)晒饣蚝哿客蛔儥z測(cè)結(jié)果提示,構(gòu)建的基因痕量突變檢測(cè)方法可以運(yùn)用于多種基因痕量突變的聯(lián)合檢測(cè),試驗(yàn)中閉管操作,在相同反應(yīng)條件下的檢測(cè)的數(shù)據(jù)結(jié)果更精確,且大大縮短反應(yīng)時(shí)間,減少實(shí)驗(yàn)成本和重復(fù)性實(shí)驗(yàn)操作,有較強(qiáng)的臨床使用價(jià)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R73-3

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