WO2010097020A1 - Braf基因突变的检测探针、液相芯片及其检测方法 - Google Patents

Braf基因突变的检测探针、液相芯片及其检测方法 Download PDF

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WO2010097020A1
WO2010097020A1 PCT/CN2010/070425 CN2010070425W WO2010097020A1 WO 2010097020 A1 WO2010097020 A1 WO 2010097020A1 CN 2010070425 W CN2010070425 W CN 2010070425W WO 2010097020 A1 WO2010097020 A1 WO 2010097020A1
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seq
mutation
braf gene
exon
pcr
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PCT/CN2010/070425
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French (fr)
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许嘉森
朱泽尧
陈玲
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广州益善生物技术有限公司
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Priority to US13/203,068 priority Critical patent/US20120053075A1/en
Priority to EP10745804A priority patent/EP2402457A4/en
Publication of WO2010097020A1 publication Critical patent/WO2010097020A1/zh

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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a detection probe for a BRAF gene mutation, a liquid phase chip and a detection method thereof. Background technique
  • the full name of the BRAF gene is the murine sarcoma virulence (v-raf) oncogene homology Bl, which is located on human chromosome 7q34. Its functional coding region consists of 2510 bases, encoding the serine sulphate in the MAPK pathway. A protein kinase that transduces signals from RAS to MEK1/2 and is involved in regulating cell growth, proliferation, and apoptosis. It is now generally accepted that the T1799A mutation in exon 15 of the BRAF gene results in the replacement of proline in the 600 residues of the BRAF protein product by glutamate (V600E), which in turn activates BRAF kinase, resulting in sustained activation of the MAPK pathway. , cells unrestricted division, added value. Other sites of mutations in the BRAF gene occur less frequently, and the BRAF mutation rate is reported to be around 15%, and more than 90% of the V600E mutations in exon 15.
  • V600E glutamate
  • the established BRAF gene mutation detection technology is mainly PCR-sequencing and real-time fluorescent quantitative PCR detection technology.
  • PCR-sequencing has the advantage of being able to determine the range and type of mutations. It is the most widely used detection method, but the sensitivity of sequencing is only 20%-25%, which is far from meeting the needs of practical applications, especially for heterogeneity. Mutations in tumor somatic cells, low sensitivity will lead to a large number of missed tests.
  • sequencing detection is complicated and time-sensitive. For clinical tests requiring high timeliness and high sensitivity, the limitations of sequencing have long been prominent.
  • the real-time fluorescence quantitative PCR detection technology can achieve a sensitivity of 1% - 2%, high detection efficiency and strong timeliness, but its false positive rate is also a problem for practical applications.
  • Luminex Corporation of the United States has developed a suspension liquid phase chip technology based on microspheres.
  • the technology utilizes polystyrene microspheres as a carrier for the reaction, and uses a fluorescence detector as a detection platform to perform high-throughput multi-index parallel detection of biological macromolecules such as nucleic acids and proteins.
  • different proportions of red light and infrared light coloring agents are incorporated to form up to 100 different color coded microspheres.
  • microspheres covalently bind protein or nucleic acid molecules for different analytes as probe molecules, reporter molecules are labeled with biotin, and stained with highly sensitive fluorescent dyes. These microspheres and the analyte, reporter, and fluorescent label form a complete microsphere detection system for reading in the Luminex system.
  • the Luminex reading system inspires the red laser and the green laser to detect the microsphere system, respectively, in which the red laser detects the intensity of the red-classified fluorescence on the surface of the microsphere, and sorts the numbers according to the different colors in the microsphere to determine the reaction.
  • Type The green laser detects the fluorescence intensity of the fluorescent marker in the sample, and then automatically counts and analyzes the type and quantity of the microspheres detected by the laser through a machine and a computer, thereby determining the respective concentrations of the plurality of target test objects of the sample to be tested. Therefore, the liquid phase chip technology not only meets the requirements of high-throughput detection, but also has the advantages of fast and accurate, high sensitivity, good specificity, and good repeatability. Summary of the invention
  • One of the objects of the present invention is to provide a probe sequence for detecting mutation of exon 15 of BRAF gene.
  • a wild type probe selected from exon 15 of any one of SEQ ID NO. 1 to SEQ ID NO. 2, and one selected from the group consisting of SEQ ID NO. 3 to SEQ ID NO.
  • Another object of the present invention is to provide a BRAF gene exon 15 mutation detection liquid phase chip.
  • a BRAF gene mutation detection liquid phase chip mainly comprises: (A) a base sequence having a base selected from any one of SEQ ID NO. 1 to SEQ ID NO. 2, for exon 15 wild type a microsphere of an amino-modified probe, and a microsphere coated with an amino-modified probe having a base sequence selected from any one of SEQ ID NO. 3 to SEQ ID NO. 4 for an exon 15 mutant, a spacer arm is attached between the base sequence of each of the above probes and the amino group, and each of the above microspheres has a different color code;
  • (B) A primer for amplifying a target sequence having a mutation site of exon 15 of the BRAF gene, the primer having an enzyme cleavage site, and the end of the amplified target sequence having a biotin label.
  • the spacer arm described above is a sequence for spacing a specific probe from the surface of the microsphere or placing the specific probe in a hydrophilic environment.
  • a spacer sequence of an appropriate length between the probe sequence and the amino group By arranging a spacer sequence of an appropriate length between the probe sequence and the amino group, steric hindrance can be reduced, the efficiency of the hybridization reaction, and the specificity of the hybridization reaction can be improved.
  • Common spacer sequences include poly dT, ie poly(dT), oligotetraethylene glycol, and (CH2)n spacer arms (n ⁇ 3), such as (CH2)12, (CH2)18, and the like.
  • poly (dA) interference is present, poly (TTG) can also be used as the spacer arm.
  • the spacer arm of the present invention is preferably 5-30! 1 .
  • the primer for amplifying the target sequence having the exon 15 mutation site comprises: comprising two rounds of PCR primers, the first round of PCR primers carrying a restriction enzyme site, and at least one of the two rounds of PCR primers Biotin label with end.
  • the first round of PCR primers are the sequences of SEQ ID NO. 5 and SEQ ID NO. 6, and the second round of PCR primers is SEQ ID NO. 5 and SEQ ID NO. 7, at least one of the primers Biotin label at the end.
  • Another object of the present invention is to provide a method for detecting mutation of exon 15 of BRAF gene, which method has rapid, Accurate, easy to operate and so on.
  • step (1) the PCR-amplified product in step (1) is digested with an endonuclease
  • the detection result is specific, the sensitivity is high, and the detection accuracy is more than 99%.
  • the detection method of the invention has simple steps, thereby avoiding many uncertain factors existing in the complicated operation process, thereby greatly improving the detection accuracy, and can be used for analyzing DNA in tissue samples and analyzing DNA in body fluids.
  • the invention adopts the enzymatic digestion enrichment method to carry out PCR amplification of the target sequence and is used for detection, thereby avoiding interference caused by a large number of wild type sequences in the product on the detection result.
  • FIG. 1 Schematic diagram of BRAF gene exon 15 wild type TspRI digestion
  • FIG. 1 Schematic diagram of BRAF gene exon 15 mutant TspRI digestion. detailed description
  • the BRAF gene mutation is a somatic mutation, and the mutation detection is interfered by a large number of wild-type genes. Therefore, in addition to the probe designed to effectively detect the BRAF gene mutation, another key question is: how to detect the nucleic acid receptor A very small number of BRAF mutant sequences that interfere with a large number of wild-type genes. How to eliminate the interference of wild-type sequences on detection is a key issue for R&D. Design two pairs of PCR primers, of which, the first round of PCR primer bands There is an endonuclease cleavage site.
  • the primer may be labeled with a biotin label at the end, or the biotin-labeled dCTP may be incorporated during the second round of the PCR reaction.
  • the primer is preferably used. Biotin label with end.
  • the invention adopts the enzymatic digestion enrichment method, firstly amplifies the wild and mutant sequences by one round of PCR, and then removes a large number of wild-type genes by enzymatic digestion, and then amplifies the mutant gene by two rounds of PCR, thereby achieving the enrichment mutant type.
  • the BRAF gene exon 15 mutant was then detected by liquid phase chip technology.
  • Washing solution 0.2ml/L Tween-20 (Sigma P-9416), lg/L SDS (Sigma L-4390)
  • oligonucleotide probes were designed for the wild type and mutant sequences of BRAF exon 15. When synthesizing the oligonucleotide probe sequence, add a 5- to 30-T spacer arm sequence (usually 10 T, the effect is consistent with 5-30 ⁇ ). The specific steps for each microsphere coating are as follows (press The conventional method is carried out):
  • microsphere mother liquor purchased from Luminex
  • the prepared BRAF gene exon 15 mutation detection liquid phase chip comprises: two kinds of microspheres coated with a wild type probe and two kinds of microspheres coated with a mutant probe, and one pair is selected when used.
  • a microsphere liquid phase chip coated with a wild type probe and a mutant probe can be used for detection.
  • microspheres coated with the probe the base sequence of each microsphere coated probe is as follows:
  • Primer a primer for amplifying a target sequence enriched with a corresponding mutation site of the exon 15 of the BRAF gene, and the end of the amplified target sequence may have a biotin label, each primer
  • the base sequence is as follows:
  • AxyPrep extraction kit to extract nucleic acids and obtain 10 samples of DNA to be detected.
  • the reaction system is as follows:
  • PCR reaction system The reaction system is sterilized per reaction (ul) ddH20 28. 8
  • Example 1 Preparation of microsphere working solution: In Example 1, microspheres coated with wild type amino modified probes of SEQ ID NO. 1 and SEQ ID NO. 2 against codon 15 of BRAF gene and coated with SEQ ID NO. 3 and SEQ ID NO. 4 microspheres for mutant amino-modified probes of BRAF gene codon 15 (wild-type amino-modified probes and mutant amino-modified probes constitute different probe sets), vortexing The microspheres were suspended, and the desired microspheres were removed into a sterile centrifuge tube (0.5 ul per reaction, wherein each microsphere was 2000/ul), and 1.5 X of the hybridization buffer was added (each reaction). 32. 5ul) and TE buffer (14ul per reaction), this is a microsphere working solution;
  • the product after the reaction was detected by a Luminex sequencer, and the results are shown in Table 1.
  • the fluorescence value (MFI) is greater than 100 as a cut-off value.
  • MFI value of the mutant probe is greater than 100, the sample is determined to be a V600E mutation in the presence of exon 15, otherwise the sample is determined to be exon 15 wild type. .
  • 2 out of 10 samples detected in this example were exon 15 point mutations (No. 3, No. 5 sample).
  • the experimental results show that the analysis results of the liquid phase chip detection are consistent with the results of the direct sequencing detection analysis, that is, there are 2 exception mutations in the sample 15 point mutation V600E (No. 3, No. 5 sample).
  • the method for detecting BRAF gene mutation using free nucleic acid provided by the present invention is feasible, and the liquid phase chip and method provided by the present invention are stable and reliable;
  • the results of the different probe sets are consistent with the results of the analysis, that is, there are 2 exceptions in the 10 samples with a point mutation of V600E (samples Nos. 3 and 5).
  • the method for detecting BRAF gene mutation in the two sets of probes provided by the present invention (SEQ ID NO. 1 and P SEQ ID NO. 3; SEQ ID NO. 2 and P SEQ ID NO. 4) is feasible, and also illustrates The probe provided by the present invention is accurate and reliable.
  • V600E point mutation V600E point mutation wild type
  • V600E point mutation V600E point mutation wild type
  • wild type wild type wild type 1 wild type wild type SEQ ID NO. 1; 2 wild type wild type
  • V600E point mutation V600E point mutation Mutant type 4 wild type wild type
  • V600E point mutation V600E point mutation wild type

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Description

BRAF基因突变的检测探针、 液相芯片及其检测方法 技术领域
本发明属于生物技术领域， 具体的是涉及 BRAF基因突变的检测探针、 液相芯片及 其检测方法。 背景技术
BRAF 基因全名为鼠类肉瘤滤过性毒菌 (v-raf) 致癌同源体 Bl， 定位于人染色体 7q34， 其具有功能的编码区由 2510对碱基组成， 编码 MAPK通路中的丝氨酸苏氨酸 蛋白激酶，该酶将信号从 RAS 转导至 MEK1/2,从而参与调节细胞的生长、增值和凋亡。 现在已普遍认同的是 BRAF基因第 15外显子上的 T1799A突变导致 BRAF蛋白产物的 600 残基中的缬氨酸被谷氨酸替代（V600E), 进而持续激活 BRAF激酶， 造成 MAPK通路持续 活化， 细胞无限制分裂、 增值。 BRAF基因突变其它位点出现的频率较低， 据报道 BRAF 突变率为 15%左右， 且 90%以上为外显子 15 的 V600E突变。
目前， 很多实验室在对 BRAF基因研究分析中， 会碰到例如检测的灵敏度和特性都 很低的困难。 已经建立的 BRAF基因突变检测技术， 主要是 PCR—测序法和实时荧光定 量 PCR检测技术。 PCR-测序法具有能够确定突变范围和类型的优点， 是目前应用最广泛 的检测方法， 但测序法的灵敏度只有 20 %— 25 %， 远远不能满足实际应用的需要， 尤 其是对于异质性的肿瘤体细胞突变， 低灵敏度将导致大量的漏检。 同时， 测序法检测操 作复杂， 时效性也差， 对于要求高时效性和高灵敏度临床检测， 测序法的局限性早已凸 显。 而实时荧光定量 PCR检测技术， 其灵敏度可以达到 1 %— 2 %， 检测效率高， 时效 性强， 但其高居不下的假阳性率也为实际应用所诟病。 基于芯片的原理， 美国 Luminex 公司开发出了以微球为载体的悬浮液相芯片技术。该项技术利用聚苯乙烯微球作为反应 的载体， 以荧光检测仪作为检测平台， 对核酸和蛋白质等生物大分子进行高通量的多指 标并行检测。在微球的制造过程中， 掺入不同比例的红光及红外光染色剂， 从而形成多 至 100种不同颜色编码的微球。不同的微球共价结合了针对不同待检测物的蛋白质或核 酸分子作为探针分子， 报告分子以生物素标记， 并用高灵敏的荧光染料染色。这些微球 与待测物、报告分子、荧光标记物就形成完整的微球检测体系用于 Luminex系统的读取。
Luminex阅读系统分别激发红色激光和绿色激光用于微球体系的检测， 其中红色激光检 测微球表面红色分类荧光的强度， 并根据微球中不同色彩而编号分类， 从而确定反应的 类型； 绿色激光检测样本中荧光标记物的荧光强度， 再通过机器与计算机自动统计分析 激光所检测到微球种类、 数量， 从而判定待测样本多种目标测试物各自的浓度。 因此， 液相芯片技术既满足了高通量检测的要求，同时具备了快速准确，灵敏度高，特异性好， 结果重复性好等优点。 发明内容
本发明的目的之一在于提供 BRAF基因外显子 15突变检测的探针序列。
用于 BRAF基因外显子 15突变检测的探针： 包括有
选自于 SEQ ID NO. 1〜SEQ ID NO. 2任一种的、 针对外显子 15的野生型探针， 和选 自于 SEQ ID NO. 3〜SEQ ID NO. 4任一种的、 针对外显子 15的突变型探针。
本发明的另一目的是提供 BRAF基因外显子 15突变检测液相芯片。
实现上述目的的技术方案如下：
一种 BRAF基因突变检测液相芯片，主要包括有： (A)包被有碱基序列选自于 SEQ ID NO. 1〜SEQ ID NO. 2任一种的、 针对外显子 15野生型的氨基修饰探针的微球， 和包被 有碱基序列选自于 SEQ ID NO. 3〜SEQ ID NO. 4任一种的、 针对外显子 15突变型的氨基 修饰探针的微球， 上述每种探针的碱基序列与氨基之间连接有间隔臂， 上述每种微球具 有不同颜色编码； 以及
(B) 用于扩增出具有 BRAF基因外显子 15突变位点的目标序列的引物， 该引物带 有酶切位点， 且扩增到的目标序列的末端具有生物素标记。
以上所述间隔臂为用于将特异性的探针与微球表面间隔开来或是将特异性探针置 于亲水性环境中的序列。通过在探针序列与氨基之间设置适当长度的间隔臂序列， 可减 少空间位阻， 提高杂交反应的效率以及杂交反应的特异性。常见的间隔臂序列包括多聚 dT， 即 poly ( dT)， 寡聚四聚乙二醇以及 (CH2) n间隔臂 (n^3), 如 (CH2) 12、 (CH2) 18等。 另外， 如果存在 poly ( dA) 干扰， 还可以用 poly (TTG) 作为间隔臂。 本发明 间隔臂优选为 5— 30个！1。
优选地，用于扩增出具有外显子 15突变位点的目标序列的引物为：包括有两轮 PCR 引物， 第一轮 PCR引物带有酶切位点， 两轮 PCR引物中至少有一条带有末端的生物素标 记。 更优选为， 第一轮 PCR引物为 SEQ ID NO. 5和 SEQ ID NO. 6序列， 第二轮 PCR引物 为 SEQ ID NO. 5和 SEQ ID NO. 7， 所述引物中至少有一条带有末端的生物素标记。 本发 明的另一目的还在于提供一种 BRAF基因外显子 15突变的检测方法， 该方法具有快速、 准确、 操作简便等优点。
一种 BRAF基因外显子 15突变的检测方法，使用上述的 BRAF基因外显子 15突变检 测液相芯片， 包括以下步骤： （1 )将待测样本 DNA用带有酶切位点的 PCR引物进行第一 轮 PCR扩增；
( 2 ) 将步骤 （1 ) 中的 PCR扩增后的产物用内切酶进行酶切；
( 3 )以酶切后的产物为模板进行对突变型的 BRAF基因进行第二轮 PCR扩增； 得到 具有外显子 15突变位点的目标 PCR扩增后产物， 且产物的末端具有生物素标记；
( 4) 第二轮 PCR扩增产物与上述相应的包被有探针的微球进行杂交；
( 5 ) 杂交反应后加入链霉亲和素-藻红蛋白进行反应， 然后进行信号检测。
本发明的主要优点在于：
( 1 ) 采用本发明提供的 BRAF基因外显子 15突变检测液相芯片， 检测结果特异性 好， 灵敏度高， 检测准确度达 99 %以上。
( 2 ) 本发明的检测方法步骤简单， 因而避免了复杂操作过程中存在的诸多不确定 因素， 因而可大大提高检测准确率， 可用于分析组织样品中的 DNA，也可分析体液中的 DNA。
( 3 ) 本发明所提供的检测方法和液相芯片所需要的检测时间远远低于常用的测序 技术。
( 4) 本发明采用酶切富集的方法进行目标序列的 PCR扩增进而用于检测， 避免了 产物中大量野生型序列对检测结果所造成的干扰。 附图说明
图 1 BRAF基因外显子 15野生型 TspRI酶切示意图；
图 2 BRAF基因外显子 15突变型 TspRI酶切示意图。 具体实施方式
BRAF基因突变是一种体细胞突变， 突变检测受大量野生型基因的干扰， 因此本发 明的除了设计到能有效检测 BRAF基因突变的探针外， 另一个关键问题是： 如何检测出 核酸中受大量野生型基因干扰的极少量 BRAF突变性序列。 如何排除野生型序列对检测 的干扰是研发首要解决的一个关键问题。 设计两对 PCR引物， 其中， 第一轮 PCR引物带 有内切酶酶切位点。为使最终扩增产物带上生物素标记， 可使引物带有末端的生物素标 记， 也可以在第二轮 PCR反应过程中掺入生物素标记的 dCTP， 本发明实施例中优选为 使引物带有末端的生物素标记。本发明采用酶切富集方法， 先用一轮 PCR同步扩增野生 和突变序列， 然后通过酶切去除大量的野生型基因， 再用二轮 PCR扩增突变型基因， 从 而达到富集突变型序列的目的， 然后用液相芯片技术检测 BRAF基因外显子 15突变型。
本发明需要用到的主要材料
溶液配方： 偶联液 （ρΗ4·5): 0. lmol/L MES (Sigma M-2933)
洗涤液： 0.2ml/L Tween-20 (Sigma P-9416), lg/L SDS (Sigma L-4390)
TE(pH8.0) (储存液）： lOmmol/L Tris( Sigma 337501), lmmol/L EDTA( Sigma E-5134) 2XTm杂交缓冲液
Figure imgf000005_0001
过滤后贮存于 4°C。 实施例 1 BRAF基因外显子 15突变检测液相芯片的制备
一、 探针序列设计及微球包被
针对 BRAF外显子 15的野生型与突变型序列， 设计特异的寡核苷酸探针。寡核苷酸 探针序列合成时， 加上一段 5— 30个 T的间隔臂序列 （通常为 10个 T， 效果与 5— 30 个 Τ一致） 每种微球包被的具体步骤如下 （按常规方法进行）：
( 1 ) 取微球母液 （购自 Luminex公司） 涡旋振荡成微球悬液；
(2) 取出 8ul微球母液， 共含 0.8X 105— 1.2X 105个微球至 0.5ml离心管中；
(3) 10， OOOrpm离心 3min， 弃去上清液；
(4) 加入 lOul偶联液 （pH4.5)， 充分混匀；
(5) 加入 2pmol/ul探针工作液 2ul;
(6) 加入 2.5ul 浓度为 5mg/ml 的 EDC (1-乙基 - (3-二甲基氨基丙基） -碳酰 二亚胺盐酸盐） 工作液， 25°C孵育 30min; 重复该步骤一次；
(7) 加入 0.2ml 洗涤液， 充分混匀， 12，000g 离心 3min， 弃去上清液； 重复该 步骤一次；
( 8 ) 加入 500ulTE溶液， 充分混匀；
( 9 ) 12，000g 离心 3min， 弃去上清液;
( 10 ) 加入 20ulTE溶液， 储存于 2-8 °C。
制备得到的 BRAF基因外显子 15突变检测液相芯片包括有：两种包被有野生型探针 的微球和两种包被有突变型探针的微球，使用时选择其中的一对包被有野生型探针和突 变型探针的微球液相芯片既可用于检测。
A. 包被探针的微球， 每种微球包被的探针的碱基序列如下:
Figure imgf000006_0001
B.引物：用于扩增出富集有 BRAF基因外显子 15需检测的相应突变位点的目标序列 的引物， 且扩增出的目标序列的末端可具有生物素标记， 每种引物的碱基序列如下:
Figure imgf000006_0002
实施例 2 运用 BRAF基因突变检测液相芯片对临床样本的检测
一、 待测样本的准备
参照 AxyPrep提取试剂盒说明提取核酸， 得到 10例待检测的 DNA样
二、 待测样品的 PCR扩增与酶切富集：
( 1 ) 第一轮 PCR扩增
PCR反应体系： 反应体系 每反应 (μΐ) 灭菌 ddH20 28.8
5 X Buffer 10
2.5mM dNTP混合液 2
MgCl225mM 5 引物 F: B15F(10uM) 1 引物 R: B15R1 (lOuM) 1
Taq 酶 （5U/ul) 0.2 模板 DNA 2 总体积 50
PCR反应条件:
Figure imgf000007_0001
(2) PCR产物 TspRI酶切:
反应体系如下：
Figure imgf000007_0002
(3) 第二轮 PCR扩增
PCR反应体系： 反应体系 每反应 (ul ) 灭菌 ddH20 28. 8
5 X Buffer 10
2. 5mM dNTP混合液 2
MgCl2 25mM 5
引物 F: B15F (10uM) 1
引物 R: B15R2-bio (10uM) 1
Taq 酶 （5U/ul ) 0. 2
模板 DNA 2
总体积 50
PCR反应条件:
Figure imgf000008_0001
三、 杂交及 Luminex 检测
( 1 ) 配制微球工作液： 将实施例 1中， 包被有 SEQ ID NO. 1和 SEQ ID NO. 2针 对 BRAF基因密码子 15的野生型氨基修饰探针的微球和包被有 SEQ ID NO. 3和 SEQ ID NO. 4针对 BRAF基因密码子 15的突变型氨基修饰探针的微球 (野生型氨基修饰探针和 突变型氨基修饰探针组成不同探针组）， 涡旋震荡成微球悬液， 随即取出所需微球到灭 菌离心管中（每反应 0. 5ul， 其中每种微球为 2000个 /ul)，加入 1. 5 X杂交液缓冲液（每 个反应 32. 5ul)和 TE缓冲液（每个反应 14ul)， 此为微球工作液；
( 2 )根据样品情况， 使用记号笔对 96孔板（杂交板）进行标记（阴性对照孔标记 为 " N")， 然后每孔加入 47ul微球工作液；
( 3 ) 各样品孔加入 3ul相应的待杂交检测样品 （第二轮 PCR产物）； "N"孔加入 3ul TE;
(4) 置杂交板于 PCR仪中， 95°C变性 3min; 55°C— 60°C杂交 15min;
( 5) 往杂交完毕的杂交板中加入 SA-PE工作液 （10ug/ml )， 每孔 25ul ;
(6) 置 PCR仪上 60°C反应 5min;
( 7) 按 Luminex仪器使用方法设置仪器参数 （每个样品读取微球 100个， 读数时 间 25s)。 将杂交板置于 Luminex仪器托盘中， 检测样品 MFI值。
四、 检测结果与数据分析
反应后产物通过 Luminex序列分析仪器检测，检测结果如表 1所示。以荧光值 (MFI) 大于 100为 cut-off 值， 当突变型探针的 MFI值大于 100时， 判定该样本为存在外显 子 15的 V600E突变， 否则判定该样本为外显子 15野生型。根据上述判定标准， 本实施 例所检测的 10份样本中， 2例存在 外显子 15点突变 （3号， 5号样本）。
实验结果显示， 液相芯片检测的分析结果与用直接测序检测分析的结果一致， 即例 样本中存在 2例外显子 15的点突变 V600E ( 3号， 5号样本）。 说明本发明所提供的用 游离核酸进行 BRAF基因突变检测的方法是可行的， 也说明本发明所提供的液相芯片和 方法是稳定可靠的；
如表 1和表 2所示， 不同探针组的检测结果和分析结果是一致的， 即 10例样本中 存在 2例外显子 15的点突变 V600E ( 3号和 5号样本）。 说明本发明所提供的两组探针 ( SEQ ID NO. 1禾 P SEQ ID NO. 3； SEQ ID NO. 2禾 P SEQ ID NO. 4) 进行 BRAF基因突变 检测的方法是可行的， 也说明本发明所提供的探针是准确可靠的。
表 1 样本的检测结果
探针组类型 序号 NO. 阴性对照 外显子 15野生型 外显子 15突变型 野生型： 1 18 1486 76
SEQ ID NO. 1； 2 14 1319 79
3 26 470 823
突变型： 4 21 1342 49
SEQ ID NO. 3 5 19 446 955
6 17 1387 65 7 24 1280 71
8 15 1367 78
9 18 1471 69
10 25 1404 53
1 20 1328 72
2 19 1395 60
3 14 450 886 野生型：
4 13 1321 38
SEQ ID NO. 2；
5 18 516 864
6 14 1378 64 突变型：
7 24 1402 84
SEQ ID NO. 4
8 15 1327 47
9 22 1396 52
10 18 1424 69
1 25 758 55
2 13 1025 67
3 26 324 785 野生型：
4 33 362 54
SEQ ID NO. 1；
5 14 424 904
6 19 387 56 突变型：
7 31 457 64
SEQ ID NO. 4
8 25 667 59
9 25 561 68
10 18 511 70 野生型： 1 30 458 49
SEQ ID NO. 2； 2 19 495 88
3 34 335 907 突变型： 4 23 451 58
SEQ ID NO. 3 5 17 321 851 6 25 1058 55
7 21 902 71
8 29 657 54
9 32 916 45
10 19 1241 59
样本 BRAF基因外显子 15突变类型分析结果
探针组类型 样本号 测序分析结果 液相芯片分析结果
1 野生型 野生型
2 野生型 野生型
3 V600E点突变 V600E点突变 野生型：
4 野生型 野生型
SEQ ID NO. 1；
5 V600E点突变 V600E点突变
6 野生型 野生型 突变型：
7 野生型 野生型
SEQ ID NO. 3
8 野生型 野生型
9 野生型 野生型
10 野生型 野生型
1 野生型 野生型
2 野生型 野生型
3 V600E点突变 V600E点突变 野生型：
4 野生型 野生型
SEQ ID NO. 2；
5 V600E点突变 V600E点突变
6 野生型 野生型 突变型：
7 野生型 野生型
SEQ ID NO. 4
8 野生型 野生型
9 野生型 野生型
10 野生型 野生型 野生型： 1 野生型 野生型 SEQ ID NO. 1； 2 野生型 野生型
3 V600E点突变 V600E点突变 突变型： 4 野生型 野生型
SEQ ID NO. 4 5 V600E点突变 V600E点突变
6 野生型 野生型
7 野生型 野生型
8 野生型 野生型
9 野生型 野生型
10 野生型 野生型
1 野生型 野生型
2 野生型 野生型
3 V600E点突变 V600E点突变 野生型：
4 野生型 野生型
SEQ ID NO. 2；
5 V600E点突变 V600E点突变
6 野生型 野生型 突变型：
7 野生型 野生型
SEQ ID NO. 3
8 野生型 野生型
9 野生型 野生型
10 野生型 野生型

Claims

权 利 要 求
1.用于 BRAF基因外显子 15突变检测的探针， 其特征是， 包括有：
选自于 SEQ ID NO. 1〜SEQ ID NO. 2任一种的、 针对外显子 15的野生型探针， 和 选自于 SEQ ID NO. 3〜SEQ ID NO. 4任一种的、 针对外显子 15的突变型探针。
2.—种 BRAF基因突变检测液相芯片， 其特征是， 主要包括有：
(A) 包被有碱基序列选自于 SEQ ID NO. 1〜SEQ ID NO. 2 任一种的、 针对外显子 15野生型的氨基修饰探针的微球， 和包被有碱基序列选自于 SEQ ID NO. 3〜SEQ ID NO. 4 任一种的、 针对外显子 15突变型的氨基修饰探针的微球， 上述每种探针的碱基序列与 氨基之间连接有间隔臂， 上述每种微球具有不同颜色编码； 以及
(B) 用于扩增出具有 BRAF基因外显子 15突变位点的目标序列的引物， 且扩增到 的目标序列的末端具有生物素标记。
3.根据权利要求 2所述的 BRAF基因突变检测液相芯片， 其特征是， （B) 中所述引物包 括有两轮 PCR引物， 第一轮 PCR引物带有酶切位点， 两轮 PCR引物中至少有一条带有末 端的生物素标记。
4.根据权利要求 3所述的 BRAF基因突变检测液相芯片， 其特征是， 所述第一轮 PCR引 物为 SEQ ID NO. 5和 SEQ ID NO. 6序列， 所述第二轮 PCR引物为 SEQ ID NO. 5和 SEQ ID NO. 7， 所述引物中至少有一条带有末端的生物素标记。
5.根据权利要求 2— 4任一项所述的 BRAF基因突变检测液相芯片， 其特征是， 所述间隔 臂为 5— 30个丁。
6.—种 BRAF基因外显子 15突变的检测方法， 其特征是， 使用权利要求 2— 5任一项所 述的 BRAF基因突变检测液相芯片， 包括以下步骤：
( 1 ) 将待测样本 DNA用带有酶切位点的 PCR引物进行第一轮 PCR扩增；
( 2 ) 将步骤 （1 ) 中的 PCR扩增后的产物用内切酶进行酶切；
( 3 )以酶切后的产物为模板进行对突变型的 BRAF基因进行第二轮 PCR扩增； 得到 具有外显子 15突变位点的目标 PCR扩增后产物， 且产物的末端具有生物素标记；
( 4) 第二轮 PCR扩增产物与所述相应的包被有探针的微球进行杂交； ( 5 ) 杂交反应后加入链霉亲和素-藻红蛋白进行反应， 然后进行信号检测。
7. 根据权利要求 6所述的 BRAF基因外显子 15突变的检测方法， 其特征是， 所述第一 轮 PCR引物为 SEQ ID NO. 5禾 P SEQ ID NO. 6序列， 第二轮 PCR引物为 SEQ ID NO. 5和 SEQ ID NO. 7， 所述引物中至少有一条带有末端的生物素标记。
8. 根据权利要求 6所述的 BRAF基因外显子 15突变的检测方法， 其特征是， 所述步骤 ( 4) 中的杂交温度为 55— 60°C。
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WO2013181125A2 (en) 2012-05-29 2013-12-05 Abbott Laboratories, Inc. Method of designing primers, method of detecting single nucleotide polymorphisms (snps), method of distinguishing snps, and related primers, detectable oligonucleotides, and kits
US10077474B2 (en) 2012-05-29 2018-09-18 Abbott Molecular, Inc. Method of designing primers, method of detecting single nucleotide polymorphisms (SNPs), method of distinguishing SNPs, and related primers, detectable oligonucleotides, and kits
EP3604552A1 (en) 2012-05-29 2020-02-05 Abbott Molecular Inc. Method of designing primers, method of detecting single nucleotide polymorphisms (snps), method of distinguishing snps, and related primers, detect able oligonucleotides, and kits

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