CN103923973A - Digital PCR platform based gene deletion mutation detection method and kit thereof - Google Patents

Digital PCR platform based gene deletion mutation detection method and kit thereof Download PDF

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CN103923973A
CN103923973A CN 201410041181 CN201410041181A CN103923973A CN 103923973 A CN103923973 A CN 103923973A CN 201410041181 CN201410041181 CN 201410041181 CN 201410041181 A CN201410041181 A CN 201410041181A CN 103923973 A CN103923973 A CN 103923973A
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pcr
gene
dna
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template
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王世亨
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上海涌泰生物医药科技有限公司
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]

Abstract

The invention relates to the field of the molecular biology, and concretely relates to gene deletion mutation detection method and a kit thereof. A PNA probe complementary to a wild non-mutation template sequence is added into a reaction system with digital PCR as a platform, and the repression effect of the PNA probe is utilized to make only deletion-mutated samples amplified; and the existence of mutant templates and the quantity and the proportion of the mutant templates are determined through fluorescence detection by utilizing a TaqMan probe as a fluorescent quantitative mark.

Description

—种基于数字PCR平台检测基因缺失突变的方法和试剂盒 - deletion mutant species based on the digital platform for PCR methods and kits for detection of gene

[0001] 技术领域 [0001] Technical Field

本发明涉及分子生物学领域和核酸检测领域,具体为检测基因缺失突变的方法。 The present invention relates to the field of molecular biology and nucleic acid detection field, particularly a method for detecting a gene deletion mutation.

背景技术 Background technique

[0002]缺失突变是基因变异中常见的类型,会给微生物、植动物的生物性状和人类的生理性状带来很多变化;包括蛋白质功能的部分或全部失活,形成无活性的片段等,也有可能改变生物体的基因调控等其他功能。 [0002] The mutation is a deletion mutation in the common type of microbes will bring many changes and biological traits of the plant physiological characters human animal; protein function include some or all of inactivation, inactive fragments, etc. is formed, there other features may alter gene regulation and other organisms. 基因突变的研究对生物的遗传、进化和改造具有重要意义。 Genetic Mutation of biological, evolution and transformation of great significance.

[0003] 目前基因变异的检测技术主要有直接测序法(亦称Sanger测序法)和ARMS法。 [0003] It mutation detection technologies include direct sequencing (also known as Sanger sequencing) and ARMS method. 现分别简介于下: About now we are in the following:

[0004] Sanger测序法,即Sanger(桑格)双脱氧链终止法是Frederick Sanger于1975年发明的。 [0004] Sanger sequencing, i.e., the Sanger (Sanger) dideoxy chain termination method was invented in Frederick Sanger 1975. 测序过程需要先做一个聚合酶链式反应(PCR)。 Sequencing process requires first a polymerase chain reaction (PCR). PCR过程中,双脱氧核糖核苷酸可能随机的被加入到正在合成中的DNA片段里。 During PCR, the double random deoxyribonucleotides may be added to the DNA fragments are in the synthesis. 由于双脱氧核糖核苷酸多脱了一个氧原子,一旦它被加入到DNA链上,这个DNA链就不能继续增加长度。 Since dideoxy ribonucleotide off one oxygen atom, once it is added to the DNA strand, the DNA strand can not continue to increase in length. 最终的结果是获得所有可能获得的、不同长度的DNA片段。 The end result is to obtain all, DNA fragments of different lengths may be obtained. 目前最普遍最先进的方法,是将双脱氧核糖核苷酸进行不同荧光标记。 The most common of the most advanced methods, is to double deoxyribonucleotides different fluorescent markers. 将PCR反应获得的总DNA通过毛细管电泳分离,跑到最末端的DNA就可以在激光的作用下发出荧光。 The total DNA obtained from the PCR reaction was separated by capillary electrophoresis, the DNA went endmost can emit fluorescence under the action of the laser. 由于ddATP,ddGTP, ddCTP, ddTTP (4种双脱氧核糖核苷酸)荧光标记不同,计算机可以自动根据颜色判断该位置上碱基究竟是A,T,G,C中的哪一个。 Depending ddATP, ddGTP, ddCTP, ddTTP (4 Species bis deoxyribonucleotides) fluorescent label, the computer can automatically determine the color bases according to the position exactly which A, T, G, C in. 直接测序法耗时长且灵敏度低,仅能检出突变比例在10%~20%以上的突变。 Direct sequencing method is time-consuming and the sensitivity is low, the proportion of mutations mutations in 10% to 20% only detected.

[0005] ARMS 法也称扩增阻碍突变系统(Amplification Refractory Mutation System,ARMS)、等位基因特性PCR (Allele Specific PCR,ASPCR)等,即等位基因特异性扩增法(Allele Specific Amplif ication, ASA)建立于1989 年,是PCR 技术应用的发展。 [0005] ARMS method is also known as the amplification refractory mutation system (Amplification Refractory Mutation System, ARMS), allele characteristics PCR (Allele Specific PCR, ASPCR) and the like, i.e., allele-specific amplification method (Allele Specific Amplif ication, ASA) was established in 1989, is the development of technology of PCR.

[0006] ARMS法主要用于对已知突变基因进行检测。 [0006] ARMS method is mainly used for detecting known mutant genes. 首先设计两个5'端引物,该引物的3'末端设计为一个与正常DNA互补,一个与突变DNA互补。 First, two design 5 'end primer, the primer 3' end of the normal design of a DNA complementary to a DNA complementary to the mutant. 对于纯合性突变,分别加入这两种引物及3'端引物进行两个平行PCR,只有与突变DNA完互补的引物才可延伸并得到PCR扩增产物。 Homozygous for the mutation, two primers were added and the 3 'end of the PCR primer two parallel, only the mutant DNA primer complementary completed before extending a PCR amplification product. 如果错配位于上游引物的3'端,则引物与模板DNA不配对,导致PCR不能延伸,因此这种方法称为ARMS,可选择性地扩增野生型或突变型基因。 If the mismatch primer is located upstream of the 3 'end of the primer to the template DNA do not match, PCR leads to not extend, so this method is called ARMS, selectively amplify the wild-type or mutant gene.

[0007] 上述现有技术存在以下问题: [0007] The above-described prior art has the following problems:

[0008] 1.均为定性检测,也就是说只能给出基因变异是否存在的结论。 [0008] 1. qualitative detection of both, that can only be given if there is genetic variation conclusions. 但无法测定携带基因变异的DNA量的比例,也就是说,无法进行定量分析检测,或者定量分析误差较大。 However, the ratio of the amount of DNA carrying the gene mutation can not be measured, i.e., quantitative analysis can not be performed, or quantitative analysis of errors.

[0009] 2.均给出模拟图结果,需要人工进行判读,判读方面相对比较主观。 [0009] FIG. 2. The simulation results are given by the need to manually perform interpretation, aspects relatively subjective interpretation. 无法直接得到数字化的客观结果。 Get objective results can not be directly digitized.

[0010] 3.灵敏度比较差,目前方法的灵敏最好为1%,无法满足某些高灵敏度的检测需求。 [0010] 3. The relatively poor sensitivity, current sensitivity of the method is preferably 1%, can not meet the needs of certain high detection sensitivity.

发明内容[0011] 本发明的目的在于提供一种基于数字PCR平台检测基因缺失突变的试剂盒及方法。 SUMMARY OF THE INVENTION [0011] The object of the present invention is to provide a kit and a method of deletion mutants based on the digital platform for PCR to detect gene.

[0012] 肽核酸(P印tide Nucleic Acid, PNA)是一类以中性酰胺键(假肽键)代替DNA中的糖-磷酸二酯键作为骨架的脱氧核糖核酸类似物,保留其中的碱基部位。 [0012] Peptide nucleic acids (P printed tide Nucleic Acid, PNA) is a class of neutral amide bond (pseudopeptide bond) instead of the DNA sugar - phosphodiester backbone as DNA analogs, which retain the base group site. PNA的特殊结构,使之可以不被蛋白酶或者核酸酶降解,有很高的稳定性,是一种非离子、非手性、不易被水解和酶切的分子。 PNA's special structure, so that it can not be degraded by proteases or nucleases, high stability, is a non-ionic, non-chiral, molecules not easily hydrolyzed and cleaved.

[0013] PNA与DNA模板结合能力高于普通寡核苷酸,可作为阻遏物置于引物前面,并与PCR引物部分重合;若PNA与模板正确配对时可阻止聚合酶链反应,野生型模板无法扩增;若发生错配(例如模板为发生突变的DNA),则结合能力迅速下降,失去阻遏作用。 [0013] PNA to DNA binding capacity than normal template oligonucleotide, can serve as a primer repressor placed in front, and partially overlap with PCR primers; polymerase chain reaction can be prevented if the PNA template correctly paired with the wild-type template can not amplification; if the mismatched (e.g. mutated template DNA) occurs, the binding capacity rapidly, loss of repression. 此时可用来扩增突变模板。 At this point mutation can be used to amplify the template.

[0014] 数字PCR是一种核酸分子绝对定量方法,基于单分子PCR方法来进行计数,主要采用当前分析化学热门研究领域的微流控或微滴化方法,将大量稀释后的核酸溶液分散至芯片的微反应器或微滴中,每个反应器的核酸模板数少于或者等于I个。 [0014] Digital PCR is a method for absolute quantification of a nucleic acid molecule, counting is performed based on single-molecule PCR method, mainly the current hot research field of analytical chemistry microfluidic droplet methods or a nucleic acid solution after the dispersion was diluted to a large number of microreactor chip or droplets, the number of nucleic acid templates in each reactor is equal to or less than I th. 这样经过PCR循环之后,有一个核酸分子模板的反应器就会给出荧光信号,没有模板的反应器就没有荧光信号。 Thus after the PCR cycle, the reactor has a template nucleic acid molecule will give a fluorescent signal of the unreacted template no fluorescent signal. 根据相对比例和反应器的体积,就可以推算出原始溶液的核酸浓度。 The relative proportions by volume of the reactor, we can calculate the nucleic acid concentration in the original solution.

[0015] 本发明以数字PCR为平台,在反应体系中加入与野生型非突变模板序列互补的PNA探针,利用PNA探针的阻遏作用,使得只有发生了缺失突变的样本才能被扩增;并且利用TaqMan探针作为荧光定量标记,根据荧光检测判断样品中是否存在突变的模板。 [0015] In the present invention, as the platform digital PCR, PNA probe was added to the wild type non-mutated sequence complementary to the template in the reaction system, using repression PNA probe so that the sample can occur only if the deletion mutation is amplified; and using quantitative TaqMan probe as a fluorescent marker, the presence or absence of mutation is determined based on the fluorescence detected in a sample template.

[0016] 本发明是一种高灵敏度的检测基因缺失突变的方法,步骤包括: [0016] The present invention is a method of detecting a gene deletion mutation with high sensitivity, comprising the step of:

[0017] 1.制备数字PCR混合液:含有待检测基因DNA模板的样品、正向和反向检测PCR扩增引物、标记TaqMan探针、PNA探针、正向和反向质控PCR引物、质控TaqMan探针以及PCR预混液混合,制备得到数字PCR混合液; [0017] 1. Preparation of digital PCR mixture: The sample to be detected containing genomic DNA template, PCR detection of forward and reverse amplification primers, labeled TaqMan probes, PNA probes, quality control of the forward and reverse PCR primers, QC probes and TaqMan PCR master mix was prepared by mixing to obtain a digital PCR mix;

[0018] 所述的正向和反向检测PCR扩增引物用于扩增含有缺失突变的基因;所述的标记TaqMan探针与突变位点下游DNA模板结合; [0018] The detection of forward and reverse primers were used to amplify the PCR amplification containing the gene deletion mutation; said TaqMan probe labeled with DNA template downstream of the mutation site binding;

[0019] 所述的质控PCR弓丨物用于扩增待检测的DNA模板上的保守序列;质控TaqMan探针与质控PCR引物所扩增的序列结合; QC bow Shu PCR was [0019] used to amplify the conserved sequence of the DNA template to be detected; the amplified quality control and quality control TaqMan probe PCR primer binding sequence;

[0020] 标记TaqMan和质控TaqMan探针是连接荧光基团和猝灭基团的核苷酸,两者的荧光基团类型不同; [0020] TaqMan labeled TaqMan probe is connected to and controls a fluorophore and quencher nucleotide groups, the different types of the two fluorophores;

[0021] 优选的,荧光基团选自6-羧基荧光素(FAM)、六氯-6-羧基荧光素(HEX)、Cy5 (美国Life Techno1gies)>Cy3 (美国Life Techno1gies)>VIC (美国Life Technologies),荧光猝灭基团选自6-羧基四甲基若丹明(TAMARA)、BHQl (美国Life Technologies),BHQ2(美国Life Technologies)或MGB (美国Life Technologies)。 [0021] Preferably, the fluorescent group is selected 6- carboxyfluorescein (the FAM), hexachloro-6-carboxy-fluorescein (HEX), Cy5 (U.S. Life Techno1gies)> Cy3 (U.S. Life Techno1gies)> VIC (U.S. Life Technologies), fluorescence quenching group is selected from 6-carboxy-tetramethylrhodamine (TAMARA), BHQl (US Life Technologies), BHQ2 (US Life Technologies) or MGB (U.S. Life Technologies).

[0022]PNA探针与野生型未突变的DNA互补。 [0022] PNA and DNA probes complementary to the wild-type unmutated.

[0023] 2.数字PCR混合液制作PCR微反应液滴,再进行PCR扩增反应;优选的,数字PCR混合液制作PCR微反应液滴的方法为:将数字PCR混合液加入微滴发生器,生成10000~20000个微反应液滴。 [0023] 2. Digital PCR mix droplets produced PCR microreactor, then a PCR amplification reaction; preferred method, digital PCR PCR microreactor by the mixture droplets to: Digital PCR mixture was added the droplet generator generating from 10,000 to 20,000 droplets microreactor.

[0024] 3.收集信号并进行结果判断:对PCR扩增反应后的产物进行信号收集,根据荧光信号的类型判断则待测样品中是否含有发生基因缺失突变的DNA模板,还可确定其中发生基因缺失突变的DNA模板的数量和含量。 [0024] 3. Collect the signal and determines the result: the reaction product after PCR amplification of signals collected, depending on the type of the fluorescent signal determined whether the test sample containing DNA gene deletion mutation occurs, which can also determine the occurrence the number and content of the gene deletion mutant DNA template. [0025] 可用QuantaSoft (Biorad)软件进行数据分析,计算样品中发生突变的拷贝数和含量,确定突变DNA样本的比例。 [0025] Available QuantaSoft (Biorad) software for data analysis, and calculating the content of mutated copy number in the sample, to determine the proportion of mutant DNA sample.

[0026] 由于质控PCR引物的作用,PCR微反应液滴中只要含有样品DNA模板,不论是否发生缺失突变,都会发生扩增反应,并且结合了质控TaqMan探针,因此会有荧光信号。 [0026] Since the effect of quality control of PCR primers, in the PCR reaction liquid as long as the micro-sample containing a template DNA, whether deletion mutation, amplification reaction occurs, and a combination of quality control TaqMan probe, so there will be a fluorescent signal. 发生了缺失突变的DNA模板,加入检测PCR扩增引物后,还结合了标记TaqMan探针。 Deletion mutation occurred in the DNA template, after addition of the detection PCR amplification primer also incorporates labeled TaqMan probe. 质控与标记TaqMan探针的荧光基团类型不同,因此根据不同的荧光信号可以分辨出样品中是否含有发生了基因缺失突变的DNA模板。 QC fluorophore labeled TaqMan probe with different types, depending on the fluorescent signal may be resolved in the DNA template a gene deletion mutation occurred in the sample composition. 再根据发生了突变的荧光信号数量以及总的荧光信号数量,可确定基因缺失突变的DNA模板含量,进行定量分析。 Then the number of fluorescent signal is mutated, and the total amount of fluorescent signal, can determine the content of template DNA gene deletion mutations, quantitative analysis.

[0027] 通过上述方法,可以检测位于基因特定范围内的缺失突变。 [0027] By the above method, a gene deletion mutation within a specific range can be detected is located.

[0028] 本发明将PNA探针用于基于数字PCR平台检测基因缺失突变以及制备基于数字PCR平台检测基因缺失突变的试剂盒,这种PNA探针与待检测的基因发生突变的野生型DNA模板互补。 Wild-type DNA template [0028] The PNA probes used in the present invention is based on the digital deletion mutants and PCR internet preparation kit for detecting gene deletion mutation detection based on the digital platform for gene PCR, this PNA probe to be detected mutations in the gene complementary. .

[0029] 本发明还将标记TaqMan探针用于基于数字PCR平台检测基因缺失突变以及制备基于数字PCR平台检测基因缺失突变的试剂盒,这种标记TaqMan探针与突变位点下游DNA模板结合。 [0029] The present invention will be labeled TaqMan probe for deletion mutation detection based on the digital platform PCR gene deletion mutation and the kit prepared based on the digital platform for the detection of gene PCR, this TaqMan probe labeled with a binding site mutant DNA template downstream.

[0030] 一种基于数字PCR平台检测基因缺失突变的试剂盒,含有以下物质中的至少一种: [0030] Digital PCR based gene deletion mutation detection kit platform, comprising at least one of the following substances:

[0031] (I)与未发生突变的野生型DNA模板互补的PNA探针; [0031] (I) a wild-type template DNA not mutated complementary PNA probes;

[0032] (2)与突变位点下游DNA模板结合的标记TaqMan探针; [0032] (2) downstream of the mutation site and the DNA binding of labeled TaqMan probe template;

[0033] (3)用于扩增待检测基因的正向和反向检测PCR扩增引物; [0033] (3) used to amplify the genes to be detected detection of forward and reverse PCR amplification primers;

[0034] (4)用于扩增待测基因所在DNA模板上保守序列的正向和反向质控PCR引物; [0034] (4) where used to amplify the gene test DNA template quality control forward and reverse PCR primers to conserved sequences;

[0035] (5)与待测基因所在DNA模板上保守序列结合的质控TaqMan探针。 [0035] (5) under test and quality control TaqMan probe where the DNA gene sequence conserved binding template.

[0036] 这种试剂盒可以用来检测基因组特定范围内的缺失突变,既可以进行定性分析,灵敏度可达1/2500,又可以进行定量分析,即使在突变样本含量极低,也具有很高的检测精度。 [0036] Such kits can be used to detect deletions within the genome of a specific range, both qualitative analysis, sensitivity of up to 1/2500, but also quantitative analysis, even in the mutant sample were very low, also has a high detection accuracy.

[0037] 与现有技术比较,本发明的优点在于: [0037] comparison with the prior art, the advantages of the present invention:

[0038] 1.结果判读方式:以前的技术方法结果的判读需要人工参与,肉眼依据相关图形作出最后的判断,这种判读的方式严重依赖经验,速度慢且很容易产生假阳性和假阴性的判读结果。 [0038] 1. interpretation of the results by: interpretation of the results of the previous technical methods require manual intervention, to the naked eye to make the final judgment in accordance with relevant graphics, this interpretation relies heavily on the experience of the way, it is slow and prone to false positives and false negatives interpretation of the results. 我们的技术方式给出的是数据信息,可以借助软件完全自动化的进行结果判读,从而加快了数据分析的速度,也减少了产生假阴性和假阳性判读的可能。 Our technical data are given by way of information, the results can be fully automated interpretation by software, which speeds up data analysis, but also reduces the possibility of false-negative and false-positive interpretation.

[0039] 2.结果描述的方式:以前的技术方式对基因变异的描述是定性的方式,也就是说,只是描述某个特异的基因变异是否存在于检测样本。 [0039] 2. Results manner described: the prior art described by way of the gene mutation is a qualitative way, that is, just describe a specific mutation is present in the test sample. 本技术方法对基因变异的描述是绝对定量的方式,可以给出样本所携带某种特异基因变异DNA的绝对数量和比例。 This mutation technique described method is absolutely quantitative manner, you may be given a certain proportion and absolute number of samples specific DNA gene mutation carried. 而且,即使在突变样本含量极低的情况下,定量分析的误差也很小。 Further, even at very low levels of mutant samples, the quantitative analysis of the error is small.

[0040] 3.检测灵敏度(数值越小灵敏度越好):以前技术方法的灵敏度一般在I % — 50%之间,而我们提出的技术方法对基因变异的检测灵敏度提升非常明显,为1/2500。 [0040] 3. Detection sensitivity (Sensitivity The smaller the value, the better): sensitivity of prior art methods generally I% - 50%, while the technique proposed method of detection sensitivity is very obvious mutation was 1 / 2500. 可以在非常高背景的野生DNA中检测出极其微量的携带基因变异的DNA。 It can detect a very small amount of DNA carrying the genetic variation in the DNA of wild very high backgrounds.

附图说明[0041] 图1为实施例1中,不同含量突变样本的荧光检测结果。 BRIEF DESCRIPTION [0041] FIG 1 of Example 1, varying amounts of sample fluorescence mutation detection results.

具体实施方式 Detailed ways

[0042] 实施例1 [0042] Example 1

[0043] (I)准备待测DNA样品:含有野生型EGFR基因的DNA、EGFR外显子19缺失的突变阳性DNA与野生型DNA混合的样本(其中突变体与野生型的含量比分别为1/1、1/100、1/1000、1/2500)。 [0043] (I) preparing a test DNA sample: DNA positive. 19 deletion mutant to wild type DNA hybrid exon containing the wild-type EGFR gene DNA, a sample EGFR (wherein the wild type and mutant content ratio was 1 / 1,1 / 100,1 / 1000,1 / 2500). DNA来源可以是血清、血浆、外周血、口腔黏膜、胸腔积液、体液或者组织等。 DNA source may be serum, plasma, peripheral blood, oral mucosa, pleural effusion, and other body fluids or tissues. 突变DNA来源于携带EGFR19突变的阳性细胞系。 DNA derived from a mutant carrying a mutation EGFR19 positive cell lines. 其中EGFR19外显子上c.2238 —c.2255位缺失突变。 Wherein the outer EGFR19 c.2238 -c.2255 position in exon deletion mutation.

[0044] (2)室温融解用于检测EGFR外显子19缺失的正向和反向检测PCR扩增引物及对应的标记TaqMan探针和PNA探针,以及正向和反向的质控PCR引物和质控TaqMan探针。 [0044] (2) at room temperature for melting the outer EGFR exon 19 deletion detecting forward and reverse primers for PCR amplification and detection of labeled PNA probes and TaqMan probes corresponding quality control as well as forward and reverse PCR QC primers and TaqMan probe.

[0045] 正向和反向PCR扩增弓丨物的序列为: [0045] The forward and reverse PCR amplification was bow Shu sequence:

[0046] Fl: 5,-GAGAAAGTTAAAATTCCCGTCGCT-3, [0046] Fl: 5, -GAGAAAGTTAAAATTCCCGTCGCT-3,

[0047] Rl:5,-ATGGACCCCCACACAGCA-3, [0047] Rl: 5, -ATGGACCCCCACACAGCA-3,

[0048] 标记TaqMan探针的序列包括荧光基团、核苷酸部分及猝灭基团,荧光基团和猝灭基团分别为FA M和MGB,核苷酸部分如SEQ ID N0.11所示,为:Pl: 5' -ACTCACATCGAGGATTTCC-3'。 Sequence [0048] TaqMan probe comprising labeled fluorophores and quencher nucleotide moiety, a fluorescent group and a quenching group MGB and FA M, respectively, as part of the nucleotide SEQ ID N0.11 as shown as: Pl: 5 '-ACTCACATCGAGGATTTCC-3'.

[0049] PNA 的序列如SEQ ID N0.16 所示,PNAl: 5' -GAATTAAGAGAAGCA-3' ; [0049] PNA sequence as shown in SEQ ID N0.16, PNAl: 5 '-GAATTAAGAGAAGCA-3';

[0050] 质控PCR引物用于扩增EGFR外显子2,正向和反向质控PCR引物的序列为: [0050] Quality Control PCR primers used to amplify EGFR exon 2, the forward and reverse sequences as PCR primers Quality Control:

[0051 ] CF2:5,-GCCAAGGCACGAGTAACAAGC-3 ' [0051] CF2: 5, -GCCAAGGCACGAGTAACAAGC-3 '

[0052] CR2:5, -CCTCCTCTGGAGGCTGAGAAAAT-3, [0052] CR2: 5, -CCTCCTCTGGAGGCTGAGAAAAT-3,

[0053] 质控TaqMan探针的荧光基团和猝灭基团分别为VIC和MGB,核苷酸部分如SEQ IDN0.26所示,质控TaqMan探针为: [0053] Quality Control fluorophore and quencher probes are TaqMan MGB and VIC, nucleotide moiety as shown in SEQ IDN0.26, quality control TaqMan probes:

[0054] CP2:VIC-ACGCAGTTGGGCACTT - MGB。 [0054] CP2: VIC-ACGCAGTTGGGCACTT - MGB.

[0055] (3)按照以下配比制备PCR反应液:2X数字PCR预混液(Biorad,#186-3022)与检测PCR扩增引物和质控PCR引物(600nM)、标记和质控TaqMan探针(300nM)以及PNA探针(300nM)与待检测DNA (IOng)配制成数字PCR混合液,用双蒸水补足至终体积为20 μ L。 [0055] (3) was prepared according to the following ratio of the PCR reaction solution: 2X digital PCR Master Mix (Biorad, # 186-3022) and detection of the PCR amplification primers and the control PCR primers (600nM), labeled TaqMan probes and QC (300 nM) and PNA probe (300 nM) to be detected DNA (IOng) formulated into digital PCR mixture, made up to a final volume of 20 μ L. with double distilled water 配制的数字PCR混合液中,正向和反向检测PCR扩增引物含量分别为600ηΜ,标记TaqMan探针含量为300nM,PNA探针含量300ηΜ,正向和反向质控PCR引物含量分别为600ηΜ,质控TaqMan探针含量为300ηΜ。 Digital PCR mix formulation, the detection of forward and reverse primers for PCR amplification were 600ηΜ content, content labeled TaqMan probe 300nM, PNA probes content 300ηΜ, quality control forward and reverse PCR primers contents were 600ηΜ , QC TaqMan probe content 300ηΜ.

[0056] (4)配制好的20 μ L数字PCR混合液加入到8_道的液滴制作板内,然后加入60 μ L液滴制作油到制作板,再放入QX200微滴发生器中,用于制备PCR微反应液滴。 [0056] (4) formulated 20 μ L PCR mix added to the digital channel 8_ droplets plate making, followed by addition of 60 μ L to produce oil droplets produced board, and then placed in a droplet generator QX200 for the preparation of micro-PCR reaction liquid.

[0057] (5)将制备好的PCR微反应液滴转移至96孔反应板,并用封板膜进行热封,生成的微反应液滴数量为10000~20000个。 [0057] (5) The prepared PCR reaction was micro-droplets transferred to a 96-well plate, and heat-sealed with an adhesive strip, the number of micro-droplets generated by the reaction from 10,000 to 20,000.

[0058] (6)将96孔PCR板放入PCR仪按照下面条件进行扩增反应: [0058] (6) into the 96-well PCR plate meter PCR amplification reaction the following conditions:

[0059] 94°C预变性5分钟;94°C变性10秒,65°C退火10秒,56°C延伸45秒,共进行32个循环,10°C终止反应。 Denaturation [0059] 94 ° C 5 minutes; 94 ° C denaturation 10 seconds, 65 ° C anneal 10 seconds, 56 ° C extend 45 seconds, a total of 32 cycles, 10 ° C the reaction was terminated.

[0060] (7)PCR扩增反应后将PCR反应板放置于PCR微反应液滴信号读取仪(QX200微滴荧光信号收集系统)中进行信号收集,结果如图1。 [0060] (7) PCR After PCR amplification reaction PCR reaction plate is placed in the microreactor droplets signal reading instrument (QX200 droplet fluorescence signal collection systems) for signal collection, the results shown in Figure 1. 用QuantaSoft (Biorad)软件进行数据分析,得出样本中突变DNA的绝对含量以及相对全部DNA的比例。 Data analysis by QuantaSoft (Biorad) software, the proportion of mutant DNA sample absolute content and relative total DNA derived.

[0061] 含有突变型DNA模板的微反应液滴中,同时出现FAM和VIC荧光信号,记为一个信号数;含未发生突变的野生型DNA模板的微反应液滴中,仅出现VIC荧光信号。 [0061] The reaction liquid containing fine mutant DNA template, simultaneous FAM and VIC fluorescence signal, referred to as a number of signals; microreactor droplets containing wild-type template DNA mutation does not occur, the fluorescence signal appears only VIC . 通过上述步骤检测,在突变样本含量1/2500的情况下也能检出突变。 By detecting the above-described step, in a case where the content of mutant sample can be detected mutation 1/2500.

[0062] 定量检测结果,突变型DNA模板含量不同的样本中,突变阳性信号数(即FAM信号数)与总信号数即总微反应液滴的数量比例如下: [0062] the quantitative test results, different mutant DNA template content in the sample, the number of positive signals mutations (i.e., the number of FAM signal) and the ratio of the number of the total number of the total signal, i.e. droplets microreactor as follows:

[0063] 1/1样品6477/13028计算值突变/野生=0.989/1,误差1.2% [0063] 6477/13028 1/1 sample Calcd mutant / wild = 0.989 / 1, the error is 1.2%

[0064] 1/100样品186/18700计算值突变/野生=0.01/1,误差0.2% [0064] 186/18700 1/100 sample Calcd mutant / wild = 0.01 / 1, the error is 0.2%

[0065] 1/1000 样品23/19854 计算值突变/ 野生=0.00116/1,误差16% [0065] 1/1000 sample 23/19854 Calcd mutant / wild = 0.00116 / 1, the error of 16%

[0066] 1/2500 样品11/19928 计算值突变/ 野生=0.00055/1,误差37% [0066] 1/2500 sample 11/19928 Calcd mutant / wild = 0.00055 / 1, the error of 37%

[0067] 突变型DNA模板与野生型DNA模板比例为1/1~1/100时,定量检测误差低于2%。 When [0067] the mutant to wild-type DNA template DNA template ratio of 1/1 ~ 1/100, quantitative detection error of less than 2%.

[0068] 选用以下各组之一的正向和反向PCR引物时,按上述步骤检测,在突变样本含量1/2500的情况下也能检出突变: [0068] The selection of one of the following groups of forward and reverse PCR primers, the detection step described above, in the case where the content of mutant sample can be detected 1/2500 mutations:

[0069] (I)正向F2:5,-GAAAGTTAAAATTCCCGTCGCTAT-3 ' [0069] (I) Forward F2: 5, -GAAAGTTAAAATTCCCGTCGCTAT-3 '

[0070]反向 R2:5,-GGACCCCCACACAGCAAA-3 ' [0070] Reverse R2: 5, -GGACCCCCACACAGCAAA-3 '

[0071 ] (2 )正向F3:5,-AAGTTAAAATTCCCGTCGCTATCA-3 ' [0071] (2) Forward F3: 5, -AAGTTAAAATTCCCGTCGCTATCA-3 '

[0072]反向 R3:5 ' -ACCCCCACACAGCAAAGC-3, [0072] Reverse R3: 5 '-ACCCCCACACAGCAAAGC-3,

[0073] (3 )正向F4:5,-GGTGAGAAAGTTAAAATTCCCGTC-3 ' [0073] (3) Forward F4: 5, -GGTGAGAAAGTTAAAATTCCCGTC-3 '

[0074]反向 R4:5 ' -CCCCACACAGCAAAGCAG-3 ' [0074] Reverse R4: 5 '-CCCCACACAGCAAAGCAG-3'

[0075] (4 )正向F5:5,-AAGGTGAGAAAGTTAAAATTCCCG-3 ' [0075] (4) Forward F5: 5, -AAGGTGAGAAAGTTAAAATTCCCG-3 '

[0076]反向 R5:5,-CCACACAGCAAAGCAGAA-3 '。 [0076] Reverse R5: 5, -CCACACAGCAAAGCAGAA-3 '.

[0077] 改用以下序列之一的PNA探针时(SEQ ID N0.17~20),按上述步骤检测,在突变样本含量1/2500的情况下也能检出突变: [0077] When switching to one of the following PNA probe sequences (SEQ ID N0.17 ~ 20), according to the above detecting step, in the case where the content of mutant sample can be detected 1/2500 mutations:

[0078] (I) PNA2:5 ' -ATTAAGAGAAGCAAC-3 ' [0078] (I) PNA2: 5 '-ATTAAGAGAAGCAAC-3'

[0079] (2 ) PNA3:5 ' -TAAGAGAAGCAACAT-3 ' [0079] (2) PNA3: 5 '-TAAGAGAAGCAACAT-3'

[0080] (3 ) PNA4:5,-AGAGAAGCAACATCT-3, [0080] (3) PNA4: 5, -AGAGAAGCAACATCT-3,

[0081 ] (4) PNA5:5' -AGAAGCAACATCTCC-3'。 [0081] (4) PNA5: 5 '-AGAAGCAACATCTCC-3'.

[0082] 当标记TaqMan探针的核苷酸部分改用以下序列之一时,在突变样本含量1/2500的情况下也能检出突变: [0082] When labeled TaqMan probes use one of the following nucleotide sequence portion, in a case where the content of mutant sample can be detected 1/2500 mutations:

[0083] (I)P2:5,-TCACATCGAGGATTTCCTT-3, [0083] (I) P2: 5, -TCACATCGAGGATTTCCTT-3,

[0084] (2 ) P3:5,-ACATCGAGGATTTCCTTGT-3, [0084] (2) P3: 5, -ACATCGAGGATTTCCTTGT-3,

[0085] (3) P4:5,-ATCGAGGATTTCCTTGTTG-3, [0085] (3) P4: 5, -ATCGAGGATTTCCTTGTTG-3,

[0086] (4 ) P5:5,-AGGATTTCCTTGTTGGCTT-3,。 [0086] (4) P5: 5, -AGGATTTCCTTGTTGGCTT-3 ,.

[0087]将突变型的DNA样品换为在EGFR19外显子上c.2238—c.2250位缺失突变的DNA,用上述方法检测,检出限也可达到1/2500,且定量分析的误差低于5%。 [0087] The mutant DNA sample transducer position error c.2238-c.2250 DNA deletion mutation, in the above-described method of detecting EGFR19 exon, the detection limit can be reached 1/2500, and quantitative analysis less than 5%.

[0088] 通过上述方法,可以检测EGFR19外显子上c.2230 一c.2260位缺失突变,检出限达到1/2500。 [0088] By the method described above can be detected EGFR19 exon c.2230 c.2260 a bit deletion mutations, the detection limit of 1/2500.

[0089] 正向和反向的质控PCR引物,以及质控TaqMan探针可改用以下序列,效果相同。 [0089] The forward and reverse PCR primers quality control, quality control, and can use the TaqMan probe sequence, the same effect.

[0090]正向质控 PCR 引物CFl: 5 ' -GTTTGCCAAGGCACGAGTAAC-3 '[0091 ]反向质控PCR 引物CRl: 5' -TCCTCTGGAGGCTGAGAAAATGA-3' [0090] Forward PCR primer QC CFl: 5 '-GTTTGCCAAGGCACGAGTAAC-3' [0091] Reverse PCR primer QC CRl: 5 '-TCCTCTGGAGGCTGAGAAAATGA-3'

[0092]质控 TaqMan 探针CP1VIC-TCACGCAGTTGGGCAC-MGB。 [0092] Quality Control TaqMan probe CP1VIC-TCACGCAGTTGGGCAC-MGB.

[0093] 使用荧光定量PCR的方法检测,检出限为1/100,而且在突变样本含量大于1%的情况下,定量分析的误差大于10%。 [0093] Using quantitative PCR detection method, a detection limit of 1/100, and in mutant sample content greater than 1%, quantitative analysis error is greater than 10%.

Claims (8)

1.一种基于数字PCR平台检测基因缺失突变的方法,其特征在于,包括如下步骤: (1)制备数字PCR混合液:含有待检测基因DNA模板的样品、正向和反向检测PCR扩增引物、标记TaqMan探针、PNA探针、正向和反向质控PCR引物、质控TaqMan探针以及PCR预混液混合,制备得到数字PCR混合液; 所述的正向和反向检测PCR扩增引物用于扩增含有缺失突变的基因;所述的标记TaqMan探针与突变位点下游DNA模板结合; 所述的质控PCR引物用于扩增待检测的DNA模板的保守序列;质控TaqMan探针与质控PCR引物所扩增的序列结合; 标记TaqMan和质控TaqMan探针是连接荧光基团和猝灭基团的核苷酸,两者的荧光基团类型不同; (2)数字PCR混合液制作PCR微反应液滴,再进行PCR扩增反应; (3)收集信号并进行结果判断:对PCR扩增反应后的产物进行信号收集,根据荧光信号的类型判断则待测样品中是否含有 1. A method for deletion mutation detection based on the digital platform PCR gene, comprising the steps of: (1) preparing a mixture of digital PCR: The sample to be detected containing genomic DNA template, forward and reverse PCR amplification detection primers, labeled TaqMan probes, PNA probes, the forward and reverse PCR primers quality control, quality control and TaqMan probe PCR master mix was prepared by mixing to obtain a digital PCR mix; detection of the forward and reverse PCR amplification primers were used to amplify by deletion mutation-containing gene; said TaqMan probe labeled with DNA template downstream of the mutation site binding; quality control PCR primers for amplification of the conserved sequence of the DNA to be detected template; QC TaqMan probe sequence of the amplified PCR primer binding and quality control; TaqMan labeled TaqMan probe is connected to and controls the nucleotide fluorophore and quencher groups, the different types of the two fluorophores; (2) digital PCR mix droplets produced PCR microreactor, then PCR amplification reaction; (3) collection signal and determines the result: the reaction product after PCR amplification of signal collection, the fluorescence signal is determined according to the type of the sample to be tested is if they contain 生基因缺失突变的DNA模板及其数量和含量。 Health gene deletion mutant DNA template and the number and content.
2.权利要求1所述基于数字PCR平台检测基因缺失突变的方法,其特征在于,所述荧光基团选自6-羧基荧光素、六氯-6-羧基荧光素、Cy5、Cy3或VIC,猝灭基团选自6-羧基四甲基若丹明、BHQl、BHQ2或MGB。 2. The method of deletion mutants based on the digital platform for PCR detection of gene according to claim 1, wherein the fluorescent group is selected from 6- carboxyfluorescein, hexachloro-6-carboxyfluorescein, fluorescein, Cy5, Cy3, or VIC, quencher groups are selected from 6-carboxy-tetramethyl-rhodamine, BHQl, BHQ2 or MGB.
3.权利要求1所述基于数字PCR平台检测基因缺失突变的方法,其特征在于,所述PNA探针与待检测的基因发生突变的野生型DNA模板互补。 The method of deletion mutants based on the digital platform for PCR detection of the gene as claimed in claim 1 or 2, characterized in that the wild-type DNA template of the gene to be detected with PNA probes complementary mutation.
4.PNA探针用于制备基于数字PCR平台检测基因缺失突变的试剂盒,其特征在于,所述PNA探针与待检测的基因发生突变的野生型DNA模板互补。 4.PNA probe kit for preparing deletion mutants based on the digital platform for PCR to detect gene, wherein said wild-type DNA template gene PNA probes to be complementary to the detection of mutations.
5.PNA探针用于基于数字PCR平台检测基因缺失突变,其特征在于,所述PNA探针与待检测的基因发生突变的野生型DNA模板互补。 5.PNA PCR probe based on the digital platform for the detection of gene deletion mutant, wherein said wild-type DNA template gene PNA probes to be complementary to the detection of mutations.
6.标记TaqMan探针用于制备基于数字PCR平台检测基因缺失突变的试剂盒,其特征在于,所述的标记TaqMan探针与突变位点下游DNA模板结合。 6. labeled TaqMan probes for the preparation of internet-based digital PCR detection kits gene deletion mutation, wherein said labeled TaqMan probe downstream of the mutation site and the DNA bound to the template.
7.标记TaqMan探针用于基于数字PCR平台检测基因缺失突变,所述的标记TaqMan探针与突变位点下游DNA模板结合。 7. labeled TaqMan probes for detection based on the digital platform for gene deletion mutant PCR, the TaqMan probe labeled with DNA template downstream of the mutation site binding.
8.一种基于数字PCR平台检测基因缺失突变的试剂盒,其特征在于,包括以下物质中的至少一种: Cl)与未发生突变的野生型DNA模板互补的PNA探针; (2)与突变位点下游DNA模板结合的标记TaqMan探针; (3)用于扩增待检测基因的正向和反向PCR扩增引物; (4)用于扩增待测基因所在DNA模板上保守序列的正向和反向质控PCR引物; (5)与待测基因所在DNA模板上保守序列结合的质控TaqMan探针。 A deletion mutation detection based on the digital platform for gene PCR kit comprising at least one of the following substances: Cl) with the wild-type template DNA not mutated complementary PNA probes; (2) mutation site downstream of a DNA template bound labeled TaqMan probe; (3) forward and reverse PCR used to amplify the gene to be detected amplification primer; (4) located on the test gene template DNA for amplification of conserved sequences quality control forward and reverse PCR primers; (5) under test and quality control TaqMan probe where the DNA gene sequence conserved binding template.
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