WO2016106643A1 - 检测非小细胞肺癌用药相关基因突变的引物及检测方法 - Google Patents

检测非小细胞肺癌用药相关基因突变的引物及检测方法 Download PDF

Info

Publication number
WO2016106643A1
WO2016106643A1 PCT/CN2014/095814 CN2014095814W WO2016106643A1 WO 2016106643 A1 WO2016106643 A1 WO 2016106643A1 CN 2014095814 W CN2014095814 W CN 2014095814W WO 2016106643 A1 WO2016106643 A1 WO 2016106643A1
Authority
WO
WIPO (PCT)
Prior art keywords
primer
lung cancer
cell lung
seq
mutation
Prior art date
Application number
PCT/CN2014/095814
Other languages
English (en)
French (fr)
Inventor
邵利彬
王晓倩
邵康
叶晓飞
安娜
王惠
钟国兴
Original Assignee
深圳华大基因股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳华大基因股份有限公司 filed Critical 深圳华大基因股份有限公司
Priority to PCT/CN2014/095814 priority Critical patent/WO2016106643A1/zh
Publication of WO2016106643A1 publication Critical patent/WO2016106643A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present application relates to the field of gene detection, and in particular to a primer for detecting mutation of a drug associated with non-small cell lung cancer, and a detection method thereof.
  • Lung cancer is the cancer that poses the greatest threat to Chinese patients.
  • the incidence rate is the highest among all malignant tumors, with an average of 600,000 new cases per year.
  • the number of lung cancer deaths is also the highest among all cancers. There are 490,000 deaths in the country, and the mortality rate has risen rapidly in recent years.
  • the mortality rate of lung cancer increased from 31/100,000 in 2005 to 2010.
  • the 78/100,000 people in the year are 50% higher than the second-ranked liver cancer.
  • the detection and treatment of lung cancer has become one of the most concerned issues in the medical field.
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • mutations in the DPYD gene are related to the efficacy of pemetrexed, and some mutations in the ERCC1 gene.
  • mutations in the rs11615 site may affect the efficacy of platinum-containing drugs. Detection of these sites may help doctors select appropriate chemotherapy drugs, thereby reducing the side effects of chemotherapy and increasing specificity.
  • TKIs Tyrosine Kinase Inhibitors
  • gefitinib gefitinib
  • Tarceva Erlotinib
  • Afatinib for the ERBB2 gene
  • the effect of using a targeted drug is closely related to the state of the target gene.
  • test kits on the market for specific genes such as the EGFR gene and the Kras gene.
  • most of these kits can only target a single gene.
  • the kit needs to be replaced.
  • the traditional detection kit can not combine the detection of multiple mutant genes into one product. In order to determine which drug is most suitable for patients, it is often necessary to perform multiple tests for different genes, which not only greatly increases the cost, but also consumes a lot of valuable treatment time.
  • the purpose of the present application is to provide a novel primer for detecting a mutation related to a drug related to non-small cell lung cancer, and a detection method and application based on the primer.
  • the present application discloses a primer for detecting a mutation related to a drug related to non-small cell lung cancer, the primer comprising 41 pairs of a specific primer set and a pair of universal primer sets, and 41 pairs of specific primer sets for detecting 12 non-small cells respectively.
  • the specific primer set of 41 mutation sites of lung cancer drug-related genes, the universal primer set is a common primer for the PCR amplification products of 41 pairs of specific primer sets;
  • the primer sequence of 41 pairs of specific primer sets is sequentially Seq ID No. 1 to Seq ID No. 82, wherein Seq ID No. 1 and Seq ID No. 2 are a primer set, Seq ID No. 3 and Seq ID No. 4 are a primer set, and so on;
  • the sequences of the groups are shown as Seq ID No. 83 and Seq ID No. 84.
  • both the upstream primer and the downstream primer have a universal sequence at the 5' end.
  • the sequence shown by Seq ID No. 85 and Seq ID No. 86 the universal sequence of the upstream primer corresponds to the upstream primer of the outer primer
  • the universal sequence of the downstream primer corresponds to the downstream primer of the outer primer; it is understood that these universal sequences are In order to facilitate the amplification of the external primers, it has nothing to do with the target sequence.
  • the universal primer that is, the outer primer, the PCR amplification target is a PCR amplification product of 41 pairs of specific primer sets, which is realized by 41 universal sequences added to the 5' end of the specific primer set; Therefore, the 3' end of the upstream and downstream primers of the outer primer correspond to the universal sequence added to the 5' end of the upstream and downstream primers of the 41 pairs of specific primer sets, respectively, as shown by Seq ID No. 85 and Seq ID No. 86. Sequence In the external primer, whether it is an upstream primer or a downstream primer, the 5' end can be added or modified in the case of ensuring PCR amplification. In one implementation of the present application, it is upstream of the external primer.
  • a primer sequence of about 10 bp was added to the 5' end of the primer.
  • a linker was added to the upstream primer of the outer primer and the 5' end of the downstream primer, respectively, to facilitate subsequent sequencing detection; it can be understood that neither index Sequences or linkers are used to facilitate subsequent analysis of the final PCR product of the four-primer method.
  • These sequences can be replaced according to different laboratory conditions, and the 3' and 41 pairs of specific primers are provided for the upstream and downstream primers of the outer primer.
  • the base is deleted at the 5' end of the upstream primer or the downstream primer of the outer primer without affecting the PCR amplification of the outer primer.
  • Substitutions, particularly the deletion or overall replacement of index sequences or connectors, are within the scope of this application.
  • Seq ID No. 85 and Seq ID No. The sequence of the forward and reverse primers shown in .86 does not require the design of more complex primers such as Seq ID No. 83 and Seq ID No. 84.
  • the present application also discloses a gene chip for detecting mutations related to drug-related diseases in non-small cell lung cancer, wherein the gene chip contains at least 41 detection sites, and 41 detection sites respectively There are 41 pairs of specific primer sets of the present application, and a universal primer set is also mixed in each detection site.
  • the gene chip of the present application actually adds a specific primer set and a universal primer set to each detection site, and each site corresponds to a specific site, that is, each site is used for a mutation. Detection; it can be understood that because this application needs to detect a total of 41 mutation sites, therefore, at least 41 detection sites are designed, but in practice detection, usually each mutation site needs to be repeated 10 times, that is, The actual gene chip contains 410 detection sites; in addition, some control detection sites need to be designed, so the number of detection sites of the gene chip can be designed according to actual experimental conditions and purposes, and is not specifically limited herein. .
  • the spotting amount of the specific primer set in the detection site is 5-15 ⁇ 10 -6 nmol
  • the spot amount of the universal primer set is 10-20 ⁇ 10 -6 nmol. It should be noted that the amount of spotting is determined based on the optimized amount of each primer in the PCR amplification of the four primers; the spot amount of the primer set contains an equal amount of the upstream primer and the downstream primer.
  • Another aspect of the present application discloses a method for detecting a mutation related to a drug related to non-small cell lung cancer, which comprises adding a DNA sample extracted from a subject to a detection site of a gene chip of the present application for PCR amplification. Then, the PCR amplification product is detected to obtain mutation information.
  • the PCR amplification product is detected, specifically comprising: purifying and recovering the PCR amplification product, and then sequencing the recovered PCR product, and determining the mutation information according to the sequencing result.
  • the present invention preferably uses sequencing to detect the PCR product.
  • the other side of the application also discloses the use of the primer or gene chip of the present application in the preparation of a reagent or device for detecting a mutation associated with a drug associated with non-small cell lung cancer.
  • the primer or the gene chip of the present application can be made into a kit for convenient use; it can also be integrated into some automated platforms or instruments specially for detecting mutations related to non-small cell lung cancer medication, especially for non- Detection of mutations in drug-related genes in small cell lung cancer; or as a detachable replacement module in an automated platform or instrumentation to achieve detection of mutations in drug-related genes specifically for non-small cell lung cancer.
  • the purification and recovery of the PCR amplification product is specifically carried out by a magnetic bead method.
  • the present application provides a corresponding kit for detecting a mutation related to a drug related to non-small cell lung cancer; the kit can directly adopt the primer or gene chip of the present application.
  • the gene chip is only a relatively simple use method of the primer of the present application; in the absence of the gene chip and related instruments, the primer of the present application can also be directly used for testing, and only a conventional PCR instrument can be used. achieve.
  • the detection primers of the 12 gene 41 mutation sites related to the existing non-small cell lung cancer drugs are designed as a whole, and based on this, a gene chip and a kit are developed, and 41 mutation positions can be realized at one time.
  • the detection of points provides an important analytical basis for the effective use of therapeutic drugs.
  • the primers of the present application can save a lot of time and cost, and lay a foundation for efficiently and quickly determining the best therapeutic drug.
  • Figure 1 is a graph showing the results of agarose gel electrophoresis of genomic DNA in the examples of the present application
  • Figure 2 is an agarose gel electrophoresis pattern of genomic DNA in another embodiment of the present application.
  • the present application develops a method for detecting multiple drug-related genes based on second-generation sequencing technology, which is directed to WaferGen's SmartChip
  • this application proposes a sequence of Seq ID No.1 to Seq ID No. 82, and detects 41 mutation sites of 12 non-small cell lung cancer drug-related genes, respectively, 41 pairs. a specific primer set; and a pair of universal primer sets corresponding to the sequences shown by Seq ID No. 83 and Seq ID No. 84; for the purpose of achieving the combined detection of the above plurality of drug-related genes.
  • the present application further makes them into gene chips to facilitate detection and use, and at the same time, is more advantageous for automation.
  • a universal sequence added at the 5' end thereof that is, a sequence such as Seq ID No. 85 and Seq ID No. 86, is used according to the present application.
  • the sequencing platform is added. It can be understood that if other sequencing platforms, such as Roche/454FLX, Illumina/Solexa Genome Analyzer, Applied Biosystems SOLID system, Life Technologies' new generation Ion Proton sequencer, and Hiseq series sequencer, are used, another segment and their respective platforms are required.
  • the corresponding universal sequence ie the need to replace the entire sequence of the sequence shown in Seq ID No. 85 and Seq ID No.
  • this example uses a four-primer scheme, in which a universal sequence is added to the outside of the conventional primer to form an internal primer, and then an external primer, that is, a universal sequence primer, is used for PCR reaction, and an index is added to the external primer.
  • the linker sequence can directly index and sequence the product during the PCR process, eliminating the need to interrupt the database.
  • this example uses the following scheme: 41 sets of primers and external primers are arranged on the chip, each group is repeated 10 times, and each set is sprayed with a set of inner primers and outer primers for detecting one site.
  • each outer primer was spotted at 8 ⁇ 10 -6 nmol
  • the inner primer was spotted at 5 ⁇ 10 -6 nmol
  • all the inner primers shared the same external primer
  • the “GTATCGTCGT” in the forward external primer sequence was a 10-bit index sequence, each chip Different indexes are needed to be merged on the machine during sequencing.
  • For the index sequence see the instructions for use of BGI-SE100. Commissioned by Wafergen to make Smartchip TE chips according to the above scheme.
  • DNA was extracted using the Qiagen DNA Kit (DNeasy Blood & Tisue Kit). After extraction, it was detected by agarose gel electrophoresis, with a bright band at 23,000 bp and no diffuse band at around 250 bp. The concentration was then checked using a Qubit fluorometer to ensure a concentration of 10-100 ng/ ⁇ L.
  • the mixed PCR reaction solution was dispensed into the customized SmartChip TE chip using the SmartChip TE Nanodispenser. After the liquid separation was completed, the surface of the chip was sealed with a matching membrane and centrifuged to prevent the liquid from overflowing.
  • the PCR reaction was carried out on a chip using a SmartChip TE PCR cycler, and the reaction conditions are shown in Table 2.
  • the product was collected using an Eppendorf plate centrifuge and purified using Agencourt Ampure XP magnetic beads to remove small fragments of non-specific amplified PCR products and primer dimers.
  • the purified DNA was sequenced on a BGISEQ-100 sequencer. See the BGISEQ-100 product description for specific procedures.
  • the BGI-PCT-V2.0 automated analysis process is used to analyze the data of the lower machine, and the pre-set mutation sites are detected and interpreted to generate an interpretation report.
  • cell lines containing specific mutations were tested and DNA extracts from Horizon's four cell lines were purchased to assess the sensitivity and specificity of this assay.
  • the mutation frequency of 8 sites was determined by Digital Droplet PCR and Sanger sequencing, and these 8 sites were in the site to be tested in this example.
  • Two of the cell lines had 5 positive sites and 3 negative sites, and the remaining 2 cell lines had 3 positive sites and 5 negative sites.
  • the inflammatory yellow (YH) cell line with 8 mutation frequencies of 0 was selected as a negative control, and the specific genes and detection methods of this example were used to detect the drug-related genes in the cells.
  • DNA extraction was carried out in accordance with step 3 above.
  • the extracted DNA sample was subjected to agarose gel electrophoresis results.
  • lane 1 is Takara's ⁇ -Hind III digest DNA Marker
  • lane 7 is Takara's DL2 000 DNA Marker
  • lanes 2 to 5 are Horizon's 4 cell lines.
  • DNA Lane 6 is the DNA of the inflammatory yellow cell line. The results showed that all samples had a bright band at 23,000 bp and no diffuse band around 250 bp, which was in line with expectations.
  • the concentration was then measured using a Qubit fluorometer at an average concentration of 82 ng/ ⁇ L, which was prepared at 10 ng/ ⁇ L for subsequent use.
  • the BGI-PCT-V2.0 automated analysis process is used to analyze the data of the lower machine, and the pre-set mutation sites are detected and interpreted to generate an interpretation report.
  • the process refer to the instructions for use.
  • the results showed that among the 40 loci of the 5 cell lines, 16 were positive and 24 were negative, which was consistent with the results of Digital Droplet PCR and Sanger sequencing, and the sensitivity and specificity were 100%. .
  • the primers and methods of this example were used to detect the drug-related genes in the lung cancer patients from the Huada Gene Sample Center. At the same time, the same DNA samples were tested using sanger sequencing and the most widely used second-generation sequencing platform, the Illumina Hiseq platform, as a control.
  • the BGI-PCT-V2.0 automated analysis process is used to analyze the data of the lower machine, and the pre-set mutation sites are detected and interpreted to generate an interpretation report.
  • the results showed that a total of 5 of the 41 loci were mutated, which was consistent with the results of the sanger sequencing method and the illumina Hiseq platform, the most widely used second-generation sequencing platform in the world.
  • this example also made the primer and the gene chip into kits for convenient use. Using the method of this example, 41 mutation sites can be detected at one time, which greatly saves detection time and cost, and provides an important basis for the treatment of non-small cell lung cancer, and lays a foundation for subsequent research on targeted drugs. .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本申请公开了一种用于检测非小细胞肺癌用药相关基因突变的引物及检测方法和应用。本申请的引物包括41对特异引物组和1对通用引物组，特异引物组为分别检测12个非小细胞肺癌用药相关基因41个突变位点的特异性引物，通用引物组为41对特异引物组扩增产物通用的外引物。

Description

检测非小细胞肺癌用药相关基因突变的引物及检测方法 技术领域
本申请涉及基因检测领域，特别是涉及一种用于非小细胞肺癌用药相关基因突变检测的引物，及其检测方法。
背景技术
当今社会，肿瘤已经成为对人类健康的一大威胁，2012年全球有1410万肿瘤新发病例，820万死亡病例。根据中国肿瘤登记中心2012年报，现在我国2010年新增肿瘤患者高达309万，死亡病例196万；平均每分钟有6人被确诊为肿瘤患者，按平均寿命74岁计算，每人的一生中有22％的机率患上肿瘤。而肺癌则是其中对中国患者威胁最大的癌症，发病率居所有恶性肿瘤的首位，平均每年有60万新发病例。相应的，肺癌死亡数也高居所有癌症之首，全国共有49万死亡病例，且死亡率在近些年快速上升，根据抽样调查，肺癌的死亡率从2005年的31/10万，增至2010年的78/10万人，比排名第二的肝癌高出50％，对肺癌的检测与治疗已成为医学界最关注的问题之一。
从组织学上，肺癌分为小细胞肺癌(small cell lung cancer，SCLC)和非小细胞肺癌(non-small cell lung cancer，NSCLC)，两种癌症在恶性程度，肿瘤特点和治疗方法上均有较大的区别。NSCLC患者占病人总数的比例较多，为80％-85％。
目前化疗仍在非小细胞肺癌治疗中占主导地位，许多与化疗药物有关的基因突变被陆续发现，比如DPYD基因上的突变与培美曲塞的药效相关，而ERCC1基因上的一些突变，如rs11615位点的突变，则会影响含铂药物的疗效，对于这些位点进行检测可以帮助医生选择合适的化疗药物，从而减少化疗的副作用，增加特异性。
另外近年来关于NSCLC靶向用药的研究已经非常深入，发现了许多有效的靶向药物，比如针对EGFR基因作用的酪氨酸激酶抑制剂(Tyrosine Kinase Inhibitors，TKIs)类药物吉非替尼(Gefitinib，商品名易瑞沙)，厄洛替尼(Erlotinib，商品名特罗凯)以及针对ERBB2基因的阿法替尼(Afatinib)等，这些新型靶向治疗药物有些已经通过FDA批准，有些则在进行临床试验，许多药物都取得了显著的疗效，对NSCLC患者病情的缓解和生存期的延长起到了重要作用。
使用靶向药物的效果与靶点基因的状态息息相关。以EGFR基因为例，若 EGFR基因第790位氨基酸发生突变由苏氨酸变为蛋氨酸，则会对吉非替尼产生耐药性，而Kras基因的第12和13位的突变则会导致患者对吉非替尼原发耐药。
现在市场上有许多针对特定基因如EGFR基因，Kras基因的检测试剂盒面世。然而这些试剂盒大多只能针对单个基因，当医生或患者需要检测其他用药相关基因时，则需要更换试剂盒。而且限于成本，传统的检测试剂盒也无法将多种突变基因的检测合为一个产品。而为了确定何种药物最适合患者，往往要进行多次针对不同基因的检测，不仅大大增加了成本，而且耗费大量的宝贵治疗时间。
发明内容
本申请的目的是提供一种新的用于非小细胞肺癌用药相关基因突变检测的引物，以及基于该引物的检测方法和应用。
本申请采用了以下技术方案：
本申请一方面公开了一种用于检测非小细胞肺癌用药相关基因突变的引物，该引物包括41对特异引物组和1对通用引物组，41对特异引物组为分别检测12个非小细胞肺癌用药相关基因的41个突变位点的特异性引物组，通用引物组为41对特异引物组的PCR扩增产物通用的外引物；41对特异引物组的引物序列依序如Seq ID No.1至Seq ID No.82所示，其中，Seq ID No.1和Seq ID No.2为一引物组，Seq ID No.3和Seq ID No.4为一引物组，以此类推；通用引物组的序列如Seq ID No.83和Seq ID No.84所示。
需要说明的是，本申请的引物是基于四引物法的PCR扩增原则进行设计的，因此，41对特异引物组中，无论是上游引物还是下游引物，在其5’端都具有一段通用序列，如Seq ID No.85和Seq ID No.86所示序列，上游引物的通用序列与外引物的上游引物对应，下游引物的通用序列与外引物的下游引物对应；可以理解，这些通用序列是为了便于外引物扩增的，与靶标序列是没有关系的，因此，在保障41对特异引物组中各条引物的3’端约20bp的序列不变的情况下，在保障PCR扩增的效率和准确性的情况下，对各条引物的5’端进行碱基的删减替换，或者对Seq ID No.85和Seq ID No.86所示序列部分进行整体替换，都在本申请的保护范围内。
还需要说明的是，通用引物，也就是外引物，其PCR扩增的对象是41对特异引物组的PCR扩增产物，是通过41对特异引物组的5’端添加的通用序列实现的；因此，外引物的上下游引物的3’端是分别与41对特异引物组上下游引物的5’端所添加的通用序列相对应的，如Seq ID No.85和Seq ID No.86所示序列； 而外引物中，无论是上游引物，还是下游引物，其5’端在保障PCR扩增的情况下都可以进行碱基的添加或修改，本申请的一种实现方式中，就在外引物的上游引物之5’端添加有约10bp的索引序列，同时，为了便于测序，还分别在外引物的上游引物和下游引物的5’端添加了接头，以方便后续的测序检测；可以理解，无论是索引序列还是接头都是为了方便后续对四引物法的最终PCR产物分析所用，这些序列可以根据不同的实验室条件进行替换，在保障外引物的上游引物和下游引物的3’端与41对特异引物组上下游引物的5’端所添加的通用序列相对应的情况下，在不影响外引物的PCR扩增的情况下，在外引物的上游引物或下游引物的5’端进行碱基的删减替换，特别是对索引序列或接头的删减或整体替换，都在本申请的保护范围内。此外，可以理解，如果仅仅是为了对特异引物的PCR扩增产物进行扩增，不考虑到建库或者针对不同芯片使用不同索引序列等问题，完全可以仅采用Seq ID No.85和Seq ID No.86所示序列正向引物和反向引物，而不需要设计更为复杂的Seq ID No.83和Seq ID No.84所示引物。
在本申请的引物的基础上，本申请还公开了一种用于检测非小细胞肺癌用药相关基因突变的基因芯片，该基因芯片中含有至少41个检测位点，41个检测位点中分别含有本申请的41对特异引物组，并且每个检测位点中还混合有通用引物组。
需要说明的是，本申请的基因芯片实际上就是在各个检测位点中分别添加了特异引物组和通用引物组，每个位点对应一个特异位点，即每个位点用于一个突变的检测；可以理解，因为本申请需要检测的突变位点共41个，因此，设计至少41个检测位点，但是，在实践检测中，通常每个突变位点需要重复10次，也就是说，实际的基因芯片中包含有410个检测位点；此外，还需要设计一些对照检测位点，因此基因芯片的检测位点个数可以根据实际的试验条件和目的而设计，在此不做具体限定。
优选的，本申请的基因芯片，其检测位点中，特异引物组的点样量为5-15×10-6nmol，通用引物组的点样量为10-20×10-6nmol。需要说明的是，点样量是基于优化好的四引物PCR扩增中，各引物的用量进行确定的；引物组的点样量中，含有等量的上游引物和下游引物。
本申请的另一面公开了一种检测非小细胞肺癌用药相关基因突变的方法，该方法包括，将提取自受检对象的DNA样品加入到本申请的基因芯片的检测位点，进行PCR扩增，然后对PCR扩增产物进行检测，获取突变信息。
优选的，对PCR扩增产物进行检测，具体包括，对PCR扩增产物进行纯化回收，然后对回收的PCR产物进行测序，根据测序结果判断突变信息。
需要说明的是，对PCR产物的检测方式包括很多种，本申请为了保障准确性，优选的，采用测序对PCR产物进行检测。
本申请的另一面还公开了本申请的引物或基因芯片在制备检测非小细胞肺癌用药相关基因突变的试剂或设备中的应用。
可以理解，本申请的引物或基因芯片可以制成试剂盒以方便使用；同样也可以将其整合到一些特别针对非小细胞肺癌用药相关基因突变检测的自动化平台或仪器设备中，特别用于非小细胞肺癌用药相关基因突变检测；或者作为自动化平台或仪器设备中的一个可拆卸替换的模块实现特别针对非小细胞肺癌用药相关基因突变的检测。
优选的，对PCR扩增产物进行纯化回收具体采用磁珠法进行。
在本申请的引物和基因芯片的基础上，本申请提供了相应的用于检测非小细胞肺癌用药相关基因突变的试剂盒；试剂盒可以直接采用本申请的引物或基因芯片。可以理解，基因芯片只是本申请的引物的一种比较简便的使用方式；在没有基因芯片及相关仪器的情况下，也可以直接采用本申请的引物进行试验，只需采用常规的PCR仪就可以实现。
本申请的有益效果在于：
本申请特别设计了现有的非小细胞肺癌用药相关的12个基因41突变位点的检测引物作为一个整体，并以此为基础研制了基因芯片和试剂盒，可以一次性实现41个突变位点的检测，为有效的使用治疗药物，提供了重要的分析依据。与现有的逐个突变位点进行检测相比，采用本申请的引物可以节省大量时间和成本，为高效快速的确定最佳治疗药物奠定了基础。
附图说明
图1：是本申请实施例中基因组DNA的琼脂糖凝胶电泳结果图；
图2：是本申请另一实施例中基因组DNA的琼脂糖凝胶电泳图。
具体实施方式
为了实现对有临床实验支持的多种用药相关基因的联合检测，必须打破原先试剂盒所依赖的qPCR等方法的限制。为此，本申请开发了一种基于二代测序技术的多种用药相关基因联合检测方法，本方法针对WaferGen公司的SmartChip
TE平台，基于华大基因的BGISEQ-100测序仪，设计了特异性的引物，该方法可同时检测12种基因的41个突变位点，48小时内即可完成，整个过程实现 自动化，仅需少量人工操作，具有操作简便，检测谱广，易于布置等特点。正是在这样一个研究框架下，本申请提出了Seq ID No.1至Seq ID No.82所示序列的，分别检测12个非小细胞肺癌用药相关基因的41个突变位点的，41对特异引物组；以及与之对应的Seq ID No.83和Seq ID No.84所示序列的1对通用引物组；以实现以上多种用药相关基因联合检测的目的。在以上41对特异引物组和1对通用引物组的基础上，本申请还进一步将其制成基因芯片，以方便检测使用，同时，也更有利于实现自动化。
需要说明的是，本申请的41对特异引物组中，在其5’端添加的一段通用序列，即如Seq ID No.85和Seq ID No.86所示序列，是依据本申请所采用的测序平台进行添加的。可以理解，如果采用其它测序平台，例如Roche/454FLX、Illumina/Solexa Genome Analyzer、Applied Biosystems SOLID system，Life Technologies公司新一代Ion Proton测序仪，以及Hiseq系列测序仪，则需要采用另外一段与各自的平台相应的通用序列，即需要对Seq ID No.85和Seq ID No.86所示序列部分进行整体替换；这种仅仅是测序接头的常规替换，在保障41对特异引物组中各条引物的3’端约20bp的序列不变的情况下，或者在保障41对特异引物组中各条引物的3’端的PCR扩增的情况下，都在本申请的保护范围内。需要补充说明的是，如果对Seq ID No.85和Seq ID No.86所示序列部分进行整体替换，则通用引物组中相应的部分也需进行适用性的改变；同样的，通用引物组中的索引序列以及通用引物组中5’段添加的接头等，也需要根据测序平台进行相应的改变，具体参考各测序平台的使用说明，在此不累述。
下面通过具体实施例和附图对本申请作进一步详细说明。以下实施例仅对本申请进行进一步说明，不应理解为对本申请的限制。
实施例
1.针对突变位点设计特异性引物
本例选择了与非小细胞肺癌用药相关的12个基因41位点，这些位点均与用药有相关性，而且都在100人以上的临床试验中取得显著效果，并得到大型医疗机构及专家的公认。本例使用Primer3和Primer premier5针对这些位点设计引物，为了更适合用于BGISEQ100测序，在设计引物的时候把扩增产物限定在200bp以内，然后使用Prime-Blast进行比对，获得41对特异引物组。配合四引物法以及后续的测序，设计了1对外引物，并且，在41对特异引物组上下游引物的5’端添加了与外引物相应的通用序列，最终的41对特异引物组，相对应的通用序列和1对外引物的序列详见表1。
表1 用药相关基因突变检测引物
Figure PCTCN2014095814-appb-000001
Figure PCTCN2014095814-appb-000002
Figure PCTCN2014095814-appb-000003
2.定制芯片
为了简化步骤，提高效率，本例使用了四引物方案，即在常规引物外侧加上通用序列构成内引物，然后与外引物即带有通用序列引物一起进行PCR反应，并且在外引物上添加索引和接头序列，可在PCR过程中直接为产物加上索引和测序接头，省去打断建库的麻烦。为了实现这一方案，本例使用如下方案：41组内引物和外引物布置在芯片上，每组重复10次，每个孔中喷涂一组内引物和外引物，用于检测一个位点，每条外引物点样8×10-6nmol，内引物点样5×10-6nmol，所有内引物共用同一外引物，正向外引物序列中“GTATCGTCGT”为10位索引序列，每张芯片需使用不同的索引，以便测序时合并上机，索引序列参见BGI-SE100的使用说明。委托wafergen公司按上述方案制成Smartchip TE芯片。
3.从样品组织提取DNA
本例使用Qiagen提取DNA试剂盒(DNeasy Blood&Tisue Kit)提取DNA。提取之后，用琼脂糖凝胶电泳方法对其进行检测，在23000bp处有亮带，且在250bp左右无弥散条带。然后使用Qubit荧光定量仪对其进行浓度检测，确保其浓度在10-100ng/μL。
4.配制PCR体系，进行目标区域扩增
采用通过质控的样本DNA，取350ng DNA作为模板，使用KAPA公司的试剂盒(KAPA2G Robust Hotstar Readymix)配置混合液，体系如下：2xPCR Master Mix 175μL、DNA 350ng，补DEPC水至350μL，即获得PCR反应液。
使用SmartChip TE Nanodispenser将混合好的PCR反应液分装至定制好的SmartChip TE芯片，分液完成后，用配套的膜封好芯片表面，并离心使液体不溢出来。使用SmartChip TE PCR cycler在芯片上进行PCR反应，反应条件如表2所示。
表2 芯片PCR反应条件
Figure PCTCN2014095814-appb-000004
Figure PCTCN2014095814-appb-000005
5.收集PCR产物并纯化：
反应完成后，使用Eppendorf公司的孔板离心机，将产物收集，并使用Agencourt公司的Ampure XP磁珠，进行纯化，去除小片段的非特异扩增PCR产物和引物二聚体。
6.使用BGISEQ-100进行上机测序：
将纯化完成的DNA，在BGISEQ-100测序仪上进行测序，具体操作步骤见BGISEQ-100产品说明。
7.分析数据得出报告
测序完成后，使用BGI-PCT-V2.0自动化分析流程对下机数据进行分析，检测预先设定的突变位点并进行解读，生成解读报告。
试验1
使用本例对含特定突变的细胞系进行检测，购买Horizon公司4个细胞系的DNA提取物，用以评估本例检测的灵敏性和特异性。这4个细胞系中，用Digital Droplet PCR和Sanger测序法确定了8个位点的突变频率，且这8个位点均在本例的待测位点中。其中2个细胞系有5个阳性位点，3个阴性位点，其余2个细胞系有3个阳性位点，5个阴性位点。另选这8个位点突变频率均为0的炎黄(YH)细胞系作为阴性对照，利用本例的特异性引物和检测方法，对细胞中用药相关基因进行检测。
(1)DNA提取
按照以上步骤3进行DNA提取。提取的DNA样品琼脂糖凝胶电泳结果如 图1所示，图中从左到右，泳道1为Takara公司的λ-Hind III digest DNA Marker，泳道7为Takara公司的DL2 000 DNA Marker，泳道2到5为Horizon公司的4个细胞系的DNA，泳道6为炎黄细胞系的DNA，结果显示，所有样品在23000bp处有亮带，且在250bp左右无弥散条带，符合预期。然后使用Qubit荧光定量仪对其进行浓度检测，平均浓度为82ng/μL，将其配制为10ng/μL，以备后续使用。
(2)按照以上步骤4-7对用药相关基因进行检测
测序完成后，使用BGI-PCT-V2.0自动化分析流程对下机数据进行分析，检测预先设定的突变位点并进行解读，生成解读报告，流程使用方法参见使用说明。检测结果表明，5种细胞系的40个位点中，有16个阳性位点，24个阴性位点，与Digital Droplet PCR和Sanger测序法的结果相符，且灵敏性和特异性均达100％。
试验2
采用本例的引物和方法对取自华大基因样本中心的肺癌患者的肿瘤组织，进行临床样本用药相关基因的检测。同时，采用sanger测序法和国际使用最广泛的二代测序平台illumina Hiseq平台对同样的DNA样品进行检测，作为对照。
(1)DNA提取
按照以上步骤3，使用Qiagen提取DNA试剂盒(DNeasy Blood&Tisue Kit)，从肿瘤组织提取DNA。提取之后，用琼脂糖凝胶电泳方法对其进行检测，在23000bp处有亮带，且在250bp左右无弥散条带，如图2所示，图中从左到右，泳道1为Takara公司的λ-Hind III digest DNA Marker，泳道3为Takara公司的DL2 000DNA Marker，泳道2为病人肿瘤组织中提取的DNA。然后使用Qubit荧光定量仪对其进行浓度检测，浓度为65ng/μL，将其配制成10ng/μL。
(2)按照以上步骤4-7对用药相关基因进行检测
测序完成后，使用BGI-PCT-V2.0自动化分析流程对下机数据进行分析，检测预先设定的突变位点并进行解读，生成解读报告。检测结果表明，41个位点中共有5个位点发生了突变，与sanger测序法和国际使用最广泛的二代测序平台illumina Hiseq平台验证的结果完全一致。
在以上研究的基础上，本例还将引物和基因芯片分别制成试剂盒，以方便使用。采用本例的方法，可以一次性检测41个突变位点，大大节省了检测时间和成本，为治疗非小细胞肺癌的用药选择提供了重要依据，也为后续的靶向药物的研究奠定了基础。
以上内容是结合具体的实施方式对本申请所作的进一步详细说明，不能认定本申请的具体实施只局限于这些说明。对于本申请所属技术领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本申请的保护范围。

Claims (10)

  1. 一种用于检测非小细胞肺癌用药相关基因突变的引物，其特征在于：所述引物包括41对特异引物组和1对通用引物组，所述41对特异引物组为分别检测12个非小细胞肺癌用药相关基因的41个突变位点的特异性引物组，所述通用引物组为41对特异引物组的PCR扩增产物通用的外引物；所述41对特异引物组的引物序列依序如Seq ID No.1至Seq ID No.82所示，其中，Seq ID No.1和Seq ID No.2为一引物组，Seq ID No.3和Seq ID No.4为一引物组，以此类推；所述通用引物组的序列如Seq ID No.83和Seq ID No.84所示。
  2. 根据权利要求1所述的引物在制备检测非小细胞肺癌用药相关基因突变的试剂或设备中的应用。
  3. 一种用于检测非小细胞肺癌用药相关基因突变的基因芯片，其特征在于：所述基因芯片中含有至少41个检测位点，41个检测位点中分别含有权利要求1所述的41对特异引物组，并且每个检测位点中还混合有权利要求1所述的通用引物组。
  4. 根据权利要求3所述的基因芯片，其特征在于：所述检测位点中，特异引物组的点样量为5-15×10-6nmol，通用引物组的点样量为10-20×10-6nmol。
  5. 根据权利要求3或4所述的基因芯片在制备检测非小细胞肺癌用药相关基因突变的试剂或设备中的应用。
  6. 一种检测非小细胞肺癌用药相关基因突变的方法，其特征在于：所述方法包括，将提取自受检对象的DNA样品加入到权利要求3或4所述的基因芯片的检测位点，进行PCR扩增，然后对PCR扩增产物进行检测，获取突变信息。
  7. 根据权利要求6所述的方法，其特征在于：所述对PCR扩增产物进行检测，具体包括，对PCR扩增产物进行纯化回收，然后对回收的PCR产物进行测序，根据测序结果判断突变信息。
  8. 根据权利要求7所述的方法，其特征在于：采用磁珠对PCR扩增产物进行纯化回收。
  9. 一种用于检测非小细胞肺癌用药相关基因突变的试剂盒，其特征在于：所述试剂盒中含有权利要求1所述的引物。
  10. 一种用于检测非小细胞肺癌用药相关基因突变的试剂盒，其特征在于：所述试剂盒中含有权利要求3或4所述的基因芯片。
PCT/CN2014/095814 2014-12-31 2014-12-31 检测非小细胞肺癌用药相关基因突变的引物及检测方法 WO2016106643A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2014/095814 WO2016106643A1 (zh) 2014-12-31 2014-12-31 检测非小细胞肺癌用药相关基因突变的引物及检测方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2014/095814 WO2016106643A1 (zh) 2014-12-31 2014-12-31 检测非小细胞肺癌用药相关基因突变的引物及检测方法

Publications (1)

Publication Number Publication Date
WO2016106643A1 true WO2016106643A1 (zh) 2016-07-07

Family

ID=56283917

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/095814 WO2016106643A1 (zh) 2014-12-31 2014-12-31 检测非小细胞肺癌用药相关基因突变的引物及检测方法

Country Status (1)

Country Link
WO (1) WO2016106643A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112037848A (zh) * 2020-09-02 2020-12-04 荣联科技集团股份有限公司 非小细胞肺癌基因变异及用药解读系统及解读方法、装置

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103562409A (zh) * 2011-05-25 2014-02-05 诺华股份有限公司 肺癌的生物标志物
CN103958695A (zh) * 2010-12-30 2014-07-30 意大利癌症研究基金会分子肿瘤学研究所(Ifom) 一种通过检测生物液体中miRNA来鉴定患早期肺癌的无症状高风险个体的方法
CN104017874A (zh) * 2014-06-02 2014-09-03 复旦大学 预测晚期非小细胞肺癌含铂化疗疗效及生存期的分子标记及试剂盒
CN104131102A (zh) * 2014-08-07 2014-11-05 马飞 一种判断nsclc患者对吉非替尼治疗反应性的试剂盒

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103958695A (zh) * 2010-12-30 2014-07-30 意大利癌症研究基金会分子肿瘤学研究所(Ifom) 一种通过检测生物液体中miRNA来鉴定患早期肺癌的无症状高风险个体的方法
CN103562409A (zh) * 2011-05-25 2014-02-05 诺华股份有限公司 肺癌的生物标志物
CN104017874A (zh) * 2014-06-02 2014-09-03 复旦大学 预测晚期非小细胞肺癌含铂化疗疗效及生存期的分子标记及试剂盒
CN104131102A (zh) * 2014-08-07 2014-11-05 马飞 一种判断nsclc患者对吉非替尼治疗反应性的试剂盒

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112037848A (zh) * 2020-09-02 2020-12-04 荣联科技集团股份有限公司 非小细胞肺癌基因变异及用药解读系统及解读方法、装置

Similar Documents

Publication Publication Date Title
Chan et al. Clinical significance of clonal hematopoiesis in the interpretation of blood liquid biopsy
CN104745679B (zh) 一种无创检测egfr基因突变的方法及试剂盒
WO2021036620A1 (zh) 一组卵巢癌预后相关基因的应用
JP7204004B2 (ja) 肺癌の複数遺伝子突然変異を一括検出する試薬キット
CN105969857A (zh) 非小细胞肺癌靶向治疗基因检测方法
CN109295230A (zh) 一种基于ctDNA的多基因联合突变检测评估肿瘤动态变化的方法
CN102776286B (zh) 用于检测ros1基因融合突变的引物、探针及检测试剂盒
CN105838777A (zh) ddPCR技术监测肺癌患者对酪氨酸激酶抑制剂继发耐药的方法
CN107513578A (zh) 一种用于肺癌驱动基因及易感基因早筛的核酸质谱检测方法
CN104017887A (zh) 人类braf基因突变检测引物对和探针及其试剂盒
CN106048008A (zh) 一种egfr基因超低频突变检测试剂盒
CN108715893B (zh) 一组与放疗引起的放射性脑损伤相关的snp标志物及其应用
CN110438206B (zh) 一组检测egfr基因19外显子缺失突变的引物、探针和试剂盒
JP2023505031A (ja) がんの分析のための方法及び組成物
CN103911439A (zh) 系统性红斑狼疮羟甲基化状态的差异表达基因的分析方法和应用
CN110885886B (zh) 一种胶质母细胞瘤鉴别诊断及胶质瘤生存预后的分型方法
WO2016106643A1 (zh) 检测非小细胞肺癌用药相关基因突变的引物及检测方法
CN114350812A (zh) 与奥西替尼耐药性相关的NSCLC-free RNA-3及其应用
CN108342488B (zh) 一种用于检测胃癌的试剂盒
WO2016106645A1 (zh) 检测结直肠癌用药相关基因突变的引物及检测方法
Lüsebrink et al. Pre-clinical validation of a next generation sequencing testing panel
WO2016106701A1 (zh) 检测非小细胞肺癌的qRT-PCR引物、探针、芯片、试剂盒、应用和方法
Barh et al. Single-Cell Omics: Volume 2: Technological Advances and Applications
Liu et al. Unraveling gene fusions for drug repositioning in high-risk neuroblastoma
CN108441560B (zh) 一种位于cep128基因的与放疗引起的放射性脑损伤相关的snp标志物及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14909452

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 24/11/2017)

122 Ep: pct application non-entry in european phase

Ref document number: 14909452

Country of ref document: EP

Kind code of ref document: A1