WO2021135072A1 - 用于检测肺癌驱动性基因突变的探针及检测试剂盒 - Google Patents
用于检测肺癌驱动性基因突变的探针及检测试剂盒 Download PDFInfo
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- the invention relates to a cancer gene detection method, in particular to a probe and a detection kit for detecting lung cancer driving gene mutations.
- the global incidence and mortality of lung cancer ranks first among all cancer types, and it is increasing year by year.
- the global incidence of lung cancer accounts for 11.6% of the total cancer population, and the mortality rate accounts for 11.6% of the total cancer population. 18.4% of the total cancer population.
- the incidence of lung cancer ranks first among all cancers, with an incidence of 787,000.
- the incidence is relatively high; among women, the incidence of lung cancer is second only to breast cancer, ranking second.
- the mortality rate of lung cancer in both men and women ranks first among cancer deaths, causing a serious burden on patients and society.
- it has been discovered that human tumors are very different.
- driver gene was introduced by Professor Wu Yilong, deputy dean of Guangdong Provincial People's Hospital at the Asian Cancer Conference in 2012. He pointed out that genes related to the occurrence and development of cancer can be called driver genes, which determine the main cause of this cancer.
- driver gene we know which drugs can be used to combat it, and then we can carry out individualized treatment. It is precisely because of this huge change in concept that genetic testing and individualized treatment of tumors have attracted more and more attention, and they have developed rapidly in just a few years.
- NGS Next Generation Sequencing
- Illumina's Hiseq, Miseq, Nextseq series and Life's Ion Torrent TM series sequencers are mainly represented by Illumina's Hiseq, Miseq, Nextseq series and Life's Ion Torrent TM series sequencers. It is widely used in biological research, prenatal diagnosis, genetic diagnosis and treatment. However, cancer patients often carry a small piece of gene change or a single base change. If you choose whole genome sequencing, it will cause a waste of cost and bring a heavier economic burden to the patient.
- the application of target sequence capture sequencing technology solves the above problems. This technology can customize the genomic region of interest into specific probes to hybridize with genomic DNA, enrich the DNA fragments of the target genomic region and then use second-generation sequencing Technology for sequencing.
- this method Compared with traditional polymerase chain reaction (PCR) and gene chips, this method has high throughput and high accuracy, and can detect known and unknown genes at one time, and can effectively detect high-frequency mutations and low-frequency or even low-frequency mutations in samples. Ultra-low frequency mutation.
- driver genes such as EGFR, KRAS, BRAF, MET, NRAS, PIK3CA, etc.
- chemotherapeutic-related genes such as TP53 and UGT1A1.
- Common targeted drugs are: EGFR target drug-gefitinib, erlotinib, icotinib, afatinib, osimertinib, AZD9291; MET target drug-Secure; BRAF target drug-Da Rafinis.
- Chemotherapy drugs such as: irinotecan, 5-FU, capecitabine, platinum drugs, etc.
- the most commonly used clinical gene mutation detection methods include Sanger sequencing technology, fluorescence in situ hybridization (FISH), amplification hindered mutation system technology (ARMS-PCR), fluorescent PCR technology, etc.
- Sanger sequencing technology as the gold standard for genetic testing, is mainly used to find specific gene mutations related to diseases. It is difficult to complete the screening of large sample cases without clear candidate genes or with a large number of candidate genes. Fluorescence quantitative PCR detection of the target gene only needs to detect whether the sample has an amplification signal, which can detect small mutations, but the throughput is low.
- ARMS-PCR technology can be used to detect known mutant genes, but it is not feasible for unknown mutant genes.
- FISH technology uses several different colors of fluorescein alone or mixed-labeled probes for in situ hybridization, it can detect multiple genes at the same time, but it is still limited in the detection of a large number of sites.
- the invention provides a probe and a detection kit for detecting lung cancer driving gene mutations. It can be combined to detect multiple mutations of lung cancer genes, which greatly saves patients' samples and waiting time for testing, provides a basis and guidance for individualized treatment of lung cancer patients, and can also monitor the efficacy and predict prognosis after treatment.
- the purpose of the present invention is to provide a probe and a detection kit for detecting lung cancer driving gene mutations, including probes for BRAF, EGFR, KRAS, MET, NRAS and PIK3CA driving gene mutations and TP53 and UGT1A1 chemotherapeutic drugs related Gene probes, through the detection of these genes to achieve individualized treatment of lung cancer and predict prognosis,
- the present invention provides a probe for detecting lung cancer driving gene mutations, including the following 6 groups of 248 probes:
- BRAF driver mutation specific probe sequence its sequence is SEQ ID NO.1 to SEQ ID NO.3;
- EGFR driver mutation specific probe sequence its sequence is SEQ ID NO.4 to SEQ ID NO.98;
- KRAS driver mutation specific probe sequence its sequence is SEQ ID NO.99 to SEQ ID NO.148;
- MET driver mutation specific probe sequence its sequence is SEQ ID NO.149 to SEQ ID NO.150;
- NRAS driver mutation specific probe sequence its sequence is SEQ ID NO.151 to SEQ ID NO.193;
- PIK3CA driver mutation specific probe sequence its sequence is SEQ ID NO.194 to SEQ ID NO.248;
- TP53 specific probe nucleotide sequence its sequence is SEQ ID NO.249 to SEQ ID NO.272;
- UGT1A1 specific probe sequence its sequence is SEQ ID NO.273 to SEQ ID NO.276.
- the medication guidance program of the detection kit includes: BRAF, EGFR, KRAS, MET, NRAS, PIK3CA driving gene mutations and TP53 and UGT1A1 chemotherapy drug-related gene mutations.
- BRAF BRAF
- EGFR BRAF inhibitor dabrafenib
- MEK inhibitor trametinib MEK inhibitor trametinib
- BRAF G466V, G469A, Y472C and other non-V600E mutations may not be sensitive to current selective BRAF inhibitors.
- Non-small cell lung cancer carrying EGFR activating mutations (such as exons 18, 19, 20 and 21) are sensitive to the first and second generation EGFR-TKIs.
- Non-small cell lung cancer with EGFR T790M mutation may be resistant to the first and second generation EGFR-TKI, but sensitive to the third generation EGFR-TKI.
- Non-small cell lung cancer carrying EGFR 20 exon insertion mutation may not be sensitive to existing EGFR-TKI.
- Lung squamous cell carcinoma carrying EGFR amplification may be more sensitive to anti-EGFR antibody combined with chemotherapy vs. chemotherapy alone.
- Non-small cell lung cancer with KRAS mutation may be resistant to current targeted therapies such as EGFR-TKI.
- Advanced non-small cell lung cancer carrying variable splicing mutations in exon 14 of MET is sensitive to MET inhibitors.
- Non-small cell lung cancer with high-level amplification of MET copy number may be sensitive to MET inhibitors.
- Those with NRAS mutations may be sensitive to MEK inhibitors.
- the rs1042522 locus of TP53 can predict the sensitivity to cisplatin, capecitabine, paclitaxel and oxaliplatin.
- rs8175347 and rs4148323 of UGT1A1 can predict the risk of side effects to irinotecan.
- the present invention also provides a detection kit for detecting lung cancer driving gene mutations, which includes the above-mentioned 6 groups of 248 probes and 2 groups of 28 probes.
- the probe provided by the present invention has high specificity, good accuracy, and the minimum detection limit can reach 0.1%. It can provide medication basis for lung cancer patients who are looking for targeted therapy drugs; it can also change the treatment plan for lung cancer patients who have severe toxic side effects and drug resistance during the treatment process.
- the form of the sample in the present invention is any form of tumor sample from which nucleic acid can be extracted.
- whole blood samples are preferred, including but not limited to tissue samples, puncture samples, serum and plasma; and tissue samples include but not limited to paraffin-embedded tissue, Fresh tissue and frozen sectioned tissue.
- genes related to lung cancer were screened from NCCN guidelines, COSMIC database, and drug-related databases.
- the specific genes are as follows: BRAF, EGFR, KRAS, MET, NRAS, PIK3CA, TP53 and UGT1A1.
- the probe sequence refers to SEQ ID NO.1-SEQ ID NO.276.
- the method of constructing a library and hybridizing and capturing is adopted to verify the probes involved in the present invention, and the specific steps are as follows:
- 1.3.2.2 Take 300ng of gDNA and add it to the interrupted tube, use 1x IDTE Buffer to make up to 80 ⁇ L, vortex to mix, and centrifuge for 1-3s.
- Agilent 2200 detects the fragment size of the broken DNA (generally 200-300bp).
- step 2.3.3 Place the 1.5mL centrifuge tube of step 2.3.2 on the magnetic stand and let it stand until the solution is completely clear. Aspirate and discard the supernatant, taking care to avoid attracting the magnetic beads.
- step 2.5.3 Place the 1.5mL centrifuge tube of step 2.5.2 on the magnetic stand, and let it stand until the solution is completely clear. Discard the supernatant, taking care to avoid attracting the magnetic beads.
- Hybridization Master Mix component Volume per reaction( ⁇ L) xGen 2X Hybridization Buffer 8.5 xGen Hybridization Buffer Enhancer 2.7 xGen Lockdown Panel or custom probes 4 Nuclease-Free Water 1.8 Total 17
- step 4.2.2 Quickly transfer all the hybridized samples to the 0.2mL PCR tube containing Bead Resuspension Mix in step 4.1.4, pipette to mix and centrifuge.
- the main quality control of the two patient samples is shown in Table 1: DNA quality evaluation and sequencing quality evaluation are both qualified, and the next step data analysis can be carried out.
- the data analysis results of lung cancer patients and standard products are as follows:
- the results of sequencing data analysis of another lung cancer patient showed that the genotype at the rs8175347 locus (*28) of the UGT1A1 gene was heterozygous, indicating that the use of irinotecan had a greater risk of side effects; the genotype at the rs1042522 locus of the TP53 gene was heterozygous.
- the combined type suggests that the sensitivity of using cisplatin, capecitabine, paclitaxel, and oxaliplatin may be low.
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Abstract
本发明公开了一种用于检测肺癌驱动性基因突变的探针及检测试剂盒,包括BRAF、EGFR、KRAS、MET、NRAS和PIK3CA驱动性基因突变的探针及TP53和UGT1A1化疗药物相关基因的探针,共计276条探针。本发明提供的探针最低检测限可达0.1%。可为寻找靶向治疗药物的肺癌患者提供用药依据;还可以为治疗过程中发生严重毒副作用、耐药等的肺癌患者更换治疗方案。
Description
本发明涉及癌症基因检测方法,尤其涉及一种用于检测肺癌驱动性基因突变的探针及检测试剂盒。
根据2018年世界癌症报告数据统计显示,全球肺癌的发病率和致死率在所有癌种中排首位,且逐年呈上升趋势,其中肺癌的全球发病率占癌症总发病人口的11.6%,致死率占癌症总发病人口的18.4%。在中国,肺癌的发病数位居所有癌症的榜首,发病数达78.7万。在男性中,发病比例较高;在女性中,肺癌的发病率仅次于乳腺癌,位居第二。但男女肺癌的死亡率均居癌症死亡榜首,对患者及社会造成了严重的负担。在临床肿瘤治疗过程中发现,人体肿瘤千差万别,即使是同一个部位的肿瘤,治疗效果和方法也应因人而异。此外,肿瘤的异质性高,即使是同一个人的同一部位的肿瘤也有可能包含了多种突变类型的肿瘤细胞,所以给临床治疗带来了很大的挑战。“驱动基因”这一名词的出现是在2012年亚洲肿瘤大会上,广东省人民医院副院长吴一龙教授提出的。他指出与癌症发生发展相关的基因可称为驱动基因,其决定了这个癌症的最主要原因。我们知道了驱动基因,就知道可以采用哪些药物来对抗它,就可以进行个体化治疗。也正是由于这种观念上的巨大改变,基因检测及肿瘤的个体化治疗越来越广泛的引起重视,短短几年之间发展飞速。
基因检测技术主要依赖于高通量测序仪,目前第二代测序(Next Generation Sequencing,NGS)平台主要以Illumina公司的Hiseq,Miseq,Nextseq系列及Life公司的Ion Torrent
TM系列测序仪为代表,已被广泛应用于生物学研究、产前诊断、基因诊断和治疗方面。然而肿瘤患者往往携带的是一小片段基因的改变或单个碱基的改变,如果选择全基因组测序会造成成本的浪费,且给患者带来更沉重的经济负担。目标序列捕获测序技术的应用解决了上述问题,该技术可将感兴趣的基因组区域定制成特异性探针与基因组DNA进行杂交,将目标基因组区域的DNA片段进行富集后再利用第二代测序技术进行测序。这种方法与传统聚合酶链式反应(PCR)及基因芯片相比,通量高,准确度高,且可一次性检测已知和未知基因,可以有效检测到样本的高频突变及低频甚至超低频突变。
随着对肺癌基因层面的深入研究,越来越多的与肺癌相关的驱动基因被挖掘,进而开发出多种靶向特异基因突变的药物。目前已知驱动基因如EGFR、KRAS、BRAF、MET、NRAS、PIK3CA等等,还有化疗药的相关基因如TP53和UGT1A1等。常见靶向药物有:EGFR靶药 -吉非替尼、厄洛替尼、埃克替尼、阿法替尼、奥希替尼、AZD9291;MET靶药-赛可瑞;BRAF靶药-达拉非尼。化疗药物如:伊立替康、5-FU、卡培他滨、铂类药物等。
目前,临床上最常用的基因突变检测方法有Sanger测序技术、荧光原位杂交技术(FISH),扩增阻碍突变***技术(ARMS-PCR)、荧光PCR技术等。Sanger测序技术作为基因检测的金标准,主要用于寻找与疾病有关的特定的基因突变,难以完成没有明确候选基因或候选基因数量较多的大样本病例的筛查。荧光定量PCR检测目的基因仅需检测样本是否具有扩增信号即可,可检测微小突变,但通量低。ARMS-PCR技术可用于对已知突变基因进行检测,对未知突变基因不可行。FISH技术虽然采用几种不同颜色的荧光素单独或者混合标记的探针进行原位杂交,能同时检测多个基因,但在进行大量位点的检测仍然有限。
本发明提供的一种用于检测肺癌驱动性基因突变的探针及检测试剂盒。可以联合检测肺癌多种基因的突变,大大节省了患者的样本和等待检测的时间,为肺癌患者的个体化治疗提供一种依据和指导,同时也可监测治疗后的疗效及预测预后。
发明内容
本发明的目的在于提供一种用于检测肺癌驱动性基因突变的探针及检测试剂盒,包括BRAF、EGFR、KRAS、MET、NRAS和PIK3CA驱动性基因突变的探针及TP53和UGT1A1化疗药物相关基因的探针,通过对这些基因的检测实现肺癌的个体化治疗和预测预后,
本发明提供的一种用于检测肺癌驱动性基因突变的探针,包括下列6组共248条探针:
BRAF驱动突变特异性探针序列:其序列为SEQ ID NO.1至SEQ ID NO.3;
EGFR驱动突变特异性探针序列:其序列为SEQ ID NO.4至SEQ ID NO.98;
KRAS驱动突变特异性探针序列:其序列为SEQ ID NO.99至SEQ ID NO.148;
MET驱动突变特异性探针序列:其序列为SEQ ID NO.149至SEQ ID NO.150;
NRAS驱动突变特异性探针序列:其序列为SEQ ID NO.151至SEQ ID NO.193;
PIK3CA驱动突变特异性探针序列:其序列为SEQ ID NO.194至SEQ ID NO.248;
还包括下列2组共28条探针:
TP53特异性探针核苷酸序列:其序列为SEQ ID NO.249至SEQ ID NO.272;
UGT1A1特异性探针序列:其序列为SEQ ID NO.273至SEQ ID NO.276。
以上特异性探针的序列具体如下:
1.BRAF驱动突变探针序列
2.EGFR驱动突变探针序列
3.KRAS驱动突变探针序列
4.MET驱动突变探针序列
5.NRAS驱动突变探针序列
6.PIK3CA驱动突变探针序列
7.TP53化疗药物基因探针
8.UGT1A1化疗药物基因探针
该检测试剂盒的用药指导方案,包括:BRAF、EGFR、KRAS、MET、NRAS、PIK3CA驱动性基因突变及TP53和UGT1A1化疗药物相关基因突变的用药指导方案。(1)携带BRAF V600E突变的非小细胞肺癌对BRAF抑制剂达拉非尼联合MEK抑制剂曲美替尼敏感。但携带BRAF G466V、G469A、Y472C等非V600E突变的非小细胞肺癌可能对当前选择性BRAF抑制剂不敏感。(2)携带EGFR活化突变(如外显子18、19、20和21)的非小细胞肺癌对第一代和第二代EGFR-TKI敏感。携带EGFR T790M突变的非小细胞肺癌可能对第一代和第二代EGFR-TKI耐药,但对第三代EGFR-TKI敏感。携带EGFR20号外显子***突变的非小细胞肺癌可能对现有EGFR-TKI都不敏感。携带EGFR扩增的肺鳞癌可能对抗EGFR抗体联合化疗VS单纯化疗更加敏感。(3)携带KRAS突变的非小细胞肺癌可能对EGFR-TKI等目前靶向治疗药物耐药。(4)携带MET14号外显子可变剪切突变的晚期非小细胞肺癌对MET抑制剂敏感。携带MET拷贝数高水平扩增的非小细胞肺癌可能对MET抑制剂敏感。(5)携带NRAS突变的可能对MEK抑制剂敏感。(6)携带PIK3CA基因突变(如外显子9和20),主要靶向药是brparlisib(BKM120)。如果PIK3CA发生激活突变,则可能对EGFR靶点、HER2靶点耐药,赫赛汀和易瑞沙、特罗凯效果欠佳。(7)TP53的rs1042522位点可预测对顺铂、卡培他滨、紫杉醇和奥沙利铂的敏感性。(8)UGT1A1的rs8175347和rs4148323可预测对伊立替康的毒副作用风险的高低。
本发明还提供一种检测肺癌驱动性基因突变的检测试剂盒,包括:上述的6组共248条探针和2组共28条探针。
本发明的有益效果是:本发明提供的探针特异性高,准确性好,最低检测限可达0.1%。可为寻找靶向治疗药物的肺癌患者提供用药依据;还可以为治疗过程中发生严重毒副作用、耐药等的肺癌患者更换治疗方案。
下面通过具体实施例对本发明进行描述,应当指出的是下面实施例只用于对本发明的进一步说明,但并不能用作对本发明保护范围的限制,本领域的技术人员应当理解,基于本发明的技术前提下,做出的改进和变型也视为本发明的保护范围。
本实施例中检测2例肺癌患者的新鲜全血样本及3种不同突变频率的标准品(0.1%、1%和5%)。本发明中样本的形式是可以提取核酸的任意形式的肿瘤样本,其中全血样本为优,包括但不限于组织样本,穿刺样本、血清和血浆;且组织样本包括但不限于石蜡包埋组织、新鲜组织和冰冻切片组织。
本实施例中,与肺癌相关的基因筛选自NCCN指南、COSMIC数据库及药物相关数据库。具体基因如下:BRAF、EGFR、KRAS、MET、NRAS、PIK3CA、TP53和UGT1A1。探针序列参考SEQ ID NO.1-SEQ ID NO.276。
本发明采用建库杂交捕获的方法对本发明所涉及的探针进行验证,具体步骤如下:
1样本的准备
1.1分离血浆和血细胞
将2例肺癌患者的新鲜全血样本分别离心3000rpm,5min,取上清至新的离心管中,再次将上清离心3000rpm,5min,取上清(血浆)保存于新的离心管中备用,做好标记(如不立即使用可放-20℃冻存)。
1.2 cfDNA的提取
严格按照磁珠法大体积游离核酸提取试剂盒说明书(天根,货号:DP710-01)从血浆中提取cfDNA,Qubit3.0检测浓度,无需进一步片段化。
1.3 gDNA的提取和片段化
1.3.1 gDNA的提取
严格按照QIAamp DNA Blood Mini Kit(QIAGEN,货号:163026054)从血细胞中提取gDNA,Qubit3.0检测浓度,琼脂糖凝胶电泳检测DNA完整性。
1.3.2 gDNA的片段化
1.3.2.1提前打开打断仪,使其温度降至4℃。
1.3.2.2取300ng的gDNA加入到打断管中,使用1x IDTE Buffer补齐至80μL,涡旋混匀,瞬时离心1-3s。
1.3.2.3设置5(run 30s,stop 30s)×3次的程序进行打断。
1.3.2.4 Agilent 2200检测打断后DNA的片段大小(一般为200-300bp)。
1.3.2.6将片段化后的产物转移至步骤1.3.2.5中的1.5mL离心管中,涡旋混匀,室温孵育5min。
1.3.2.7将步骤1.3.2.6的1.5mL离心管置于磁力架,静置直至溶液完全澄清,吸弃上清,注意避免吸到磁珠。
1.3.2.8加入200μL新鲜配制的80%乙醇,室温孵育30sec,吸弃上清。
1.3.2.9重复1.3.2.8。
1.3.2.10将步骤1.3.2.9的1.5mL离心管微离心后静置于磁力架,吸弃残留溶液,开盖室温晾干直至乙醇完全挥发。
1.3.2.11加入52μL NFW,涡旋混匀,室温孵育2min,置于磁力架,待溶液完全澄清后转移50μL上清至新的已标记好的0.2mL PCR管进入下一步反应。
1.3.2.12取1μL用Qubit3.0检测浓度。
2文库构建
2.1末端修复和3'端加“A”
2.1.1取30ng cfDNA/40ng标准品,用NFW补齐至50μL,涡旋混匀并离心。
2.1.2在0.2mL PCR Tube中配制以下反应体系:
组分 | 体积(μL) |
cfDNA/gDNA | 50 |
End Repair&A-Tailing Buffer | 7 |
End Repair&A-Tailing Enzyme Mix | 3 |
总体积 | 60 |
2.1.3涡旋混匀,离心后进行以下反应:
2.2加Adapter
2.2.1将上一步反应产物先离心,然后配制以下反应体系:
组分 | 体积(μL) |
End Repair and A-Tailing Reaction Product | 60 |
NFW | 5 |
Ligation Buffer | 30 |
Duplex Seq Adapters(3μM) | 5 |
DNA Ligase | 10 |
总体积 | 110 |
2.2.2涡旋混匀,离心后进行以下反应:
步骤 | 反应温度 | 反应时间 |
Adapter Ligation | 20℃ | 15min |
Hold | 4℃ | ∞ |
2.3连接后纯化
2.3.2将接头连接后的产物转移至步骤2.3.1中的1.5mL离心管中,涡旋混匀,室温孵育5min。
2.3.3将步骤2.3.2的1.5mL离心管置于磁力架,静置直至溶液完全澄清,吸弃上清,注意避免吸到磁珠。
2.3.4加入200μL新鲜配制的80%乙醇,室温孵育30sec,吸弃上清。
2.3.5重复2.3.4。
2.3.6将步骤2.3.5的1.5mL离心管微离心后静置于磁力架,吸弃残留溶液,开盖室温晾干直至乙醇完全挥发。
2.3.7加入22μL NFW,涡旋混匀,室温孵育2min,置于磁力架,待溶液完全澄清后转移20μL上清至新的已标记好的0.2mL PCR管。
2.4 PCR扩增
2.4.1在0.2mL PCR Tube中,配制以下反应体系:
组分 | 体积(μL) |
2X KAPA HiFi HotStart ReadyMix | 25 |
UDI Primer Mix(5μM/Primer) | 5 |
Adapter-ligated Library | 20 |
总体积 | 50 |
2.4.2涡旋离心,离心后进行以下反应
2.5 PCR产物纯化
2.5.2将PCR产物转移至步骤2.5.1中的1.5mL离心管中,涡旋混匀,室温孵育5min。
2.5.3将步骤2.5.2的1.5mL离心管置于磁力架,静置直至溶液完全澄清吸弃上清,注意避免吸到磁珠。
2.5.4加入200μL新鲜配制的80%乙醇,室温孵育30sec,吸弃上清。
2.5.5重复2.5.4。
2.5.6将步骤2.5.5中的1.5mL离心管微离心后静置于磁力架,吸弃残留溶液,开盖室温晾干直至乙醇完全挥发。
2.5.7加入22μL NFW,涡旋混匀,室温孵育2min,置于磁力架,待溶液完全澄清后转移20μL上清至新的已标记好的1.5mL离心管中。
2.5.8质检
取1μL DNA文库溶液用Qubit荧光定量仪检测浓度。
3杂交
3.1在1.5ml EP管中配置如下反应:
Blocker component | Volume per reaction(μL) |
Human Cot DNA | 5 |
xGen Blocking Oligos based on your library adapters | 2 |
3.2混合文库2ug,加至步骤3.1的混合液中。
3.3真空抽干。
3.4配置下列体系于已真空干燥的样本EP管中
Hybridization Master Mix component | Volume per reaction(μL) |
xGen 2X Hybridization Buffer | 8.5 |
xGen Hybridization Buffer Enhancer | 2.7 |
xGen Lockdown Panel or custom probes | 4 |
Nuclease-Free Water | 1.8 |
Total | 17 |
3.5振荡,离心,重复两次。
3.6进行以下PCR反应程序:
4捕获。
按照下表配置清洗buffer:
按照下表配置Bead Resuspension Mix
Bead Resuspension Mix component | Volume per reaction(μL) |
xGen 2X Hybridization Buffer | 8.5 |
xGen Hybridization Buffer Enhancer | 2.7 |
Nuclease-Free Water | 5.8 |
Total | 17 |
4.1清洗链霉亲和素磁珠
4.1.1振荡混匀磁珠,按每个捕获50μL吸取磁珠至同一1.5mL EP管中。
4.1.2按每个捕获加入100μL Bead Wash Buffer,用枪吹打10次以上,放于磁力架静置,直至完全澄清,弃去上清。
4.1.3重复4.1.2操作两次。
4.1.4加入已配置的Bead Resuspension Mix,按照每个捕获17μL,吹打混匀,移至0.2mL PCR管中。
4.2捕获
4.2.1探针杂交结束后,迅速将梯度PCR仪程序调至下表
4.2.2迅速将杂交后的样本全部转入步骤4.1.4中的含有Bead Resuspension Mix的0.2mL PCR管中,吹打混匀并离心。
4.2.3将样本放入步骤4.2.1已设置好程序的PCR仪中,放置45min,每隔10-12min,取出快速振荡混匀,确保磁珠处于重悬状态。
4.3热洗
4.3.1恒温仪65℃预热1X Wash Buffer 1和1X Stringent Wash Buffer至少15min。
4.3.2加入100μL预热的1X Wash Buffer 1至4.2.3的样本中,用枪快速吹打混匀,避免产生气泡。
4.3.3置于磁力架至澄清,弃上清,移除磁力架。
4.3.4加入150μL预热的1X Stringent Wash Buffer至样本,用枪快速吹打混匀,避免产生气泡。65℃孵育5min,置于磁力架至澄清,弃上清。
4.3.5重复步骤4.3.4。
4.4室温洗
4.4.1向热洗步骤4.3.5中的样本加入150μL Wash Buffer 1,振荡离心。室温孵育2min,每30s振荡一次(不要太剧烈),确保磁珠处于重悬状态。
4.4.2孵育结束后,微离心。置于磁力架1min,至澄清,弃上清。
4.4.3加入150μL Wash Buffer 2,振荡混匀。室温孵育2min,每30s振荡一次(不要太剧烈),确保磁珠处于重悬状态。
4.4.4孵育结束后,微离心。置于磁力架1min,至澄清,弃上清。
4.4.5加入150μL Wash Buffer 3,振荡混匀。室温孵育2min,30s振荡一次(不要太剧烈),确保磁珠处于重悬状态。
4.4.6孵育结束后,微离心。置于磁力架1min,至澄清,弃上清。
4.4.7用枪吸弃残余的Wash Buffer 3,移除磁力架。
4.4.8每个捕获加入20μL NFW,用枪吹打混匀,并转移至0.2mL PCR管中。
5 PCR扩增和纯化
5.1 PCR扩增
5.1.1在各个捕获中加入如下体系
Amplification Reaction Mix component | Volume per reaction(μL) |
2X KAPA HiFi HotStart ReadyMix* | 25 |
KAPA Primer mix | 5 |
5.1.2进行如下的PCR扩增
5.2 PCR产物纯化
5.2.1提前配置好80%乙醇,每个捕获需要250μL。
5.2.3加入125μL 80%乙醇,室温静置1min,弃上清。
5.2.4重复步骤5.2.3。
5.2.5室温干燥磁珠,大约1-3min。
5.2.6待磁珠干燥完毕,移除磁力架,加入22μL TE,吹打混匀。
5.2.7室温孵育5min。置磁力架至澄清,转移20μL至新的1.5mL EP管中保存。
5.2.8文库质检
使用Qubit 3.0和Agilent 2200进行检测。
6.Nextseq上机测序。
7.数据分析结果
两位患者样本的主要质量控制如表1所示:DNA质量评估和测序质量评估均合格,可进行下一步数据分析。肺癌患者和标准品的数据分析结果如下:
(1)一位肺癌患者的测序数据分析结果显示:(1)检测到EGFR基因Exon 20存在c.2294T>G突变,导致所编码第765位上的缬氨酸(V)变为甘氨酸(G)。目前尚无专门针对该变异位点的药物,有被FDA或cFDA批准的该基因靶向药物。(2)UGT1A1基因的rs8175347位点基因型为杂合型,提示使用伊立替康的毒副作用风险较大(中性粒细胞减少症,腹泻,无力)。
另外一位肺癌患者的测序数据分析结果显示:UGT1A1基因rs8175347位点(*28)基因 型为杂合型,提示使用伊立替康的毒副作用风险较大;TP53基因的rs1042522位点基因型为杂合型,提示使用顺铂、卡培他滨、紫杉醇、奥沙利铂的敏感性可能较低。
(2)三种标准品的检测结果如表2所示:等位基因频率分别为0.1%、1%和5%的已知突变位点均被检测出来。说明该检测试剂盒可以检测超低频突变的基因位点,最低检测限可达0.1%。
表1:
表2:
基因 | 已知突变位点 | 预期的等位基因频率(%) | 检测的等位基因频率(%) |
EGFR | T790M | 5.0 | 4.7 |
EGFR | delE746-A750 | 5.0 | 1.8 |
EGFR | L858R | 5.0 | 3.9 |
EGFR | V769-D770insASV | 5.0 | 3.0 |
PIK3CA | E545K | 6.3 | 5.9 |
KRAS | G12D | 6.3 | 5.0 |
EGFR | T790M | 1.0 | 0.52 |
EGFR | delE746-A750 | 1.0 | 0.19 |
EGFR | L858R | 1.0 | 0.58 |
EGFR | V769-D770insASV | 1.0 | 0.39 |
PIK3CA | E545K | 1.3 | 0.25 |
KRAS | G12D | 1.3 | 0.25 |
EGFR | T790M | 0.1 | 0.0021 |
Claims (2)
- 一种用于检测肺癌驱动性基因突变的探针,其特征在于,包括下列6组共248条探针:BRAF驱动突变特异性探针序列:其序列为SEQ ID NO.1至SEQ ID NO.3;EGFR驱动突变特异性探针序列:其序列为SEQ ID NO.4至SEQ ID NO.98;KRAS驱动突变特异性探针序列:其序列为SEQ ID NO.99至SEQ ID NO.148;MET驱动突变特异性探针序列:其序列为SEQ ID NO.149至SEQ ID NO.150;NRAS驱动突变特异性探针序列:其序列为SEQ ID NO.151至SEQ ID NO.193;PIK3CA驱动突变特异性探针序列:其序列为SEQ ID NO.194至SEQ ID NO.248;还包括下列2组共28条探针:TP53特异性探针核苷酸序列:其序列为SEQ ID NO.249至SEQ ID NO.272;UGT1A1特异性探针序列:其序列为SEQ ID NO.273至SEQ ID NO.276。
- 一种检测肺癌驱动性基因突变的检测试剂盒,包括:权利要求1所述的6组共248条探针和2组共28条探针。
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KRIEGSHÄUSER GERNOT, FABJANI GERHILD, ZIEGLER BARBARA, ZÖCHBAUER-MÜLLER SABINE, END ADELHEID, ZEILLINGER ROBERT: "Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 12, no. 12, 1 January 2011 (2011-01-01), pages 8530 - 8538, XP055826006, DOI: 10.3390/ijms12128530 * |
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