WO2017114009A1 - Egfr基因检测探针及其制备方法和试剂盒 - Google Patents

Egfr基因检测探针及其制备方法和试剂盒 Download PDF

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WO2017114009A1
WO2017114009A1 PCT/CN2016/105716 CN2016105716W WO2017114009A1 WO 2017114009 A1 WO2017114009 A1 WO 2017114009A1 CN 2016105716 W CN2016105716 W CN 2016105716W WO 2017114009 A1 WO2017114009 A1 WO 2017114009A1
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egfr gene
probe
egfr
detection
fluorescein
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陈绍宇
何瑰
欧焕金
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广州安必平医药科技股份有限公司
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  • the invention belongs to the biotechnology, in particular to an EGFR gene detection probe, a preparation method thereof and a kit.
  • Lung cancer is currently a research hotspot, but the detection hotspots mainly focus on the detection of EGFR gene mutation sites, which has potential value in small molecule TKI targeted therapy.
  • the detection hotspots mainly focus on the detection of EGFR gene mutation sites, which has potential value in small molecule TKI targeted therapy.
  • macromolecular monoclonal antibody drugs such as cetuximab
  • FISH detection of EGFR copy number changes is more valuable.
  • Current clinical studies have also found that EGFR mutations and EGFR amplification are two overlapping and independent targets. Evaluation of EGFR amplification can effectively predict the overall survival of patients and is also of positive clinical significance.
  • Fluorescence in situ hybridization is a non-radioactive in situ hybridization technique developed on the basis of the original radioactive in situ hybridization technique in the late 1980s. At present, this technology has been widely used in animal and plant genomic structure research, chromosome fine structure variation analysis, viral infection analysis, human prenatal diagnosis, tumor genetics and genome evolution research in many fields.
  • the basic principle of FISH is to use a known labeled nucleic acid as a probe to heterologously bind to an unknown single-stranded nucleic acid in a material to be tested according to the principle of base complementation to form a hybrid double-stranded nucleic acid which can be detected.
  • the probe can be directly hybridized to the chromosome to localize the specific gene on the chromosome.
  • fluorescence in situ hybridization has the characteristics of rapid detection signal, high hybridization specificity and multi-staining, so it has received widespread attention in the field of molecular cytogenetics.
  • the probes used for hybridization can be roughly classified into three categories: 1) chromosome-specific repeat probes, such as alpha satellites, satellite class III probes, which often have a hybrid target of more than 1 Mb, do not contain scattered repeats, and bind tightly to the target. Strong hybridization signal, easy to detect; 2) whole chromosome or chromosomal region-specific probe consisting of a very different nucleotide fragment on a chromosome or a segment of a chromosome, which can be cloned into phage and plasmid A chromosome-specific large fragment is obtained; 3) a specific position probe consisting of one or several cloned sequences.
  • the fluorescein labeling of the probe can be performed by direct and indirect labeling.
  • the indirect labeling is a biotin-labeled DNA probe, which is detected by fluorescein avidin or streptavidin after hybridization, and the avidin-biotin-fluorescein complex can also be used to fluoresce signals. Amplification is performed so that a fragment of about 500 bp can be detected.
  • the direct labeling method is to directly bind fluorescein to the probe nucleotide or the pentose phosphate backbone, or to incorporate fluorescein nucleoside triphosphate in the nick translation labeling probe.
  • the direct labeling method has simple steps in detection and is convenient for clinical use.
  • One of the objects of the present invention is to provide an EGFR gene detecting probe and a preparation method thereof, and the prepared probe can be used for detecting the EGFR gene state, that is, detecting the finger copy number change of the EGFR gene, and has good specificity.
  • a method for preparing an EGFR gene detecting probe comprises the following steps:
  • a BAC clone as at least one of RP11-748O6, RP11-164G7, RP11-781C22, and RP11-17J15, or selecting a BAC clone as at least one of CTD-2086F7, RP11-381G5, and CTD-2343B15;
  • the plasmid DNA is labeled with fluorescein, and the fluorescein labeled by the plasmid DNA of different sources is the same, that is, it is obtained.
  • the BAC clones are RP11-748O6, RP11-164G7, RP11-781C22, and RP11-17J15.
  • the BAC clones are CTD-2086F7, RP11-381G5, and CTD-2343B15.
  • the labeled fluorescein selects a fluorescent dye known in the art, preferably fluorescein is Alexa FITC, Alexa Rhodamine, Texas Red, pacific DEAC.
  • the labeling of the gene probe can be performed by labeling the corresponding fluorescein to the double-stranded nucleic acid by methods in the prior art, including but not limited to: random primer method, nick translation, etc., marker gene probe
  • the needle can be a commercially available nick translation labeling kit and/or a random primer labeling kit, preferably abbott and/or Roche's Nick Translation Kit.
  • the plasmid DNA is preferably subjected to fluorescein labeling by a random primer method or a nick translation method.
  • Another object of the present invention is to provide an EGFR gene detecting kit.
  • An EGFR gene detection kit comprising the above EGFR gene detection probe.
  • a chromosome 7 discrimination probe (CSP7) probe for internal control is included, the identification probe being different in color from the fluorescein labeled by the EGFR gene detection probe.
  • the invention adopts FISH (Fluorescence In-Situ Hybridization) method to detect EGFR gene copy number by screening the optimal EGFR gene detection probe and its combination, and the signal counting is accurate and rapid, and the result is reproducible; It supplements the deficiency of EGFR mutation detection in clinical, which is helpful for screening more patients who benefit from targeted drugs and improving the survival rate and overall survival of lung cancer patients.
  • FISH Fluorescence In-Situ Hybridization
  • the EGFR state change is known from the gene level, and various signal types exhibit tumor cell genetic diversity of the solid tissue, which can be applied in the fields of tumor biology and cytogenetics. It can help comprehensively evaluate each molecular marker and assist in the selection of clinical targeted therapy drugs and treatment options for lung cancer.
  • Figure 1 is a schematic illustration of the detection probe sequence in Example 1.
  • Fig. 2 is a graph showing the results of FISH detection of human peripheral blood cultured cell sheets in Example 1.
  • Fig. 3 is a diagram showing the expression pattern of the EGFR gene detecting gene amplification in Example 3.
  • Example 4 is a diagram showing the results of FISH detection of a lung cancer tissue sample in Example 4, wherein the detection signal type is 2R2G, and the EGFR gene is not amplified.
  • Fig. 5 is a graph showing the results of FISH detection of a lung cancer tissue sample in Example 4, wherein the detection signal type is 6-12R2G, and the EGFR gene is amplified.
  • GSP EGFR includes two groups, including probe set 1: first probe, second probe, third probe and fourth probe; probe set 2: first probe, second probe and third Probes, specific to the following table, were purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries. The following two sets of detection probes were separately prepared.
  • EGFR chr7 55, 086, 725-55, 275, 031, 188, 307 bp
  • Probe set 1 BAC Insert start and end position First probe RP11-748O6 Chr7:54,711,888-54,884,289,172,402bp Second probe RP11-164G7 Chr7:54,818,676-54,995,526,176,851bp Third probe RP11-781C22 Chr7: 54,987,970-55,164,028,176,059bp Fourth probe RP11-17J15 Chr7: 55, 131, 101-55, 279, 524, 148, 424 bp
  • the plasmid DNA mixture was fluorescently labeled by a nick translation method, and each probe labeled fluorescein was Spectrum-Orange.
  • the PCR reaction system was prepared on ice under strict light conditions as follows.
  • the labeled product was subjected to ethanol precipitation and concentration, and sequentially added in a 1.5 ml centrifuge tube as follows. Sodium acetate and absolute ethanol, protected from light and ice:
  • chromosome 7p11 shows a red fluorescent signal
  • CSP7 a chromosome 7 identification probe, which can be labeled with chromosome 7 and purchased from KREATECH-KBI20007
  • the EGFR gene detection kit includes two components of an EGFR hybridization solution and a DAPI counterstaining agent, wherein the EGFR hybridization solution comprises the GSP EGFR gene probe described in Example 1 (two sets of detection probes respectively, corresponding to two kits) ), CSP7 probe (Chromosome 7 identification probe), buffer component for hybridization environment (promoting hybridization), COT Human DNA blocking the repeat sequence, and the like.
  • DAPI counterstaining agent is mainly used for counterstaining of cells after hybridization, in which DAPI binds to DNA, so that the nucleus shows blue fluorescence, and the counterstaining agent containing p-phenylenediamine can maintain fluorescence stability.
  • the reaction time of pepsin needs to be determined by preliminary tests. Samples prepared in the same batch can be pre-tested as described, usually at intervals of 5 minutes. For example, the digestion time is 5 minutes, 10 minutes, and 15 minutes, respectively. After the "slide pretreatment" is completed, the tissue digestion state can be observed under a bright field using a 10 ⁇ or 20 ⁇ objective lens; or DAPI counterstaining can be directly performed. The digestion state is judged.
  • the above listed reagents were all prepared in a circular dyeing tank (40 ml each), and up to 5 slices per dyeing tank. For non-room temperature solutions, pre-heat the reagents to the specified temperature before starting the operation. During the washing process, the dyeing tank can be gently shaken at intervals of 2 to 3 minutes to improve the washing effect.
  • the relevant fluorescence and DAPI need to be observed with a suitable filter block.
  • the CSP7 probe displays a green signal;
  • the GSP EGFR probe is a red signal.
  • GSP EGFR (red) and CSP 7 (green) signals were counted in 50 tumor cells in 5 clear tumor regions, and the number of GSP EGFR and CSP 7 signals in individual nuclei were counted.
  • the signals are connected as a signal when they are connected in a line or adjacent (the pitch is smaller than the diameter of the signal point).
  • the maximum number of recorded signals is 15, and when >15, the record is 15.
  • GSP EGFR/CSP7 red/green
  • CSP7 signal in the nucleus used for counting is ⁇ 2;
  • condition a, or b, or c, or d is satisfied, it is determined to be FISH positive (EGFR gene amplification);
  • EGFR gene amplification is diverse in non-small cell lung cancer samples. Based on FISH detection, it is often expressed in six signal forms: in about 60% of the nucleus, the EGFR signal appears as a relatively loose, more than 20 copies of the signal. Type, the amplicon behaves as a homogeneously stained region (Fig. 3a); the EGFR signal appears as a 4-10 copy of the gene signal in 10% to 15% of the nucleus (Fig. 3b); the amplicon in the 15%-20% nucleus Contains the CSP7 sequence, which is expressed as a co-amplified gene cluster (Fig. 3c); approximately 5% of the nuclei have a large and bright gene signal cluster in the atypical relative internal control cells (Fig. 3d); Microbodies (Fig. 3e); chromosomal aneuploidy occurred in 5% of the nucleus, with ⁇ 15 EGFR signal appearing in ⁇ 10% of the nucleus (Fig. 3f).
  • Example 2 Using the two sets of detection probes described in Example 1, the test kit described in Example 2 was tested on 20 clinical samples (which were confirmed by pathological examination, see the table below). According to the detection method of Example 3, the detection was repeated 3 times, the results were consistent, and the repeatability of the detection results was good; the detection consistency of the two probe combinations was good. Compared with commercially available commercial reagents, the test results are completely consistent, and the specificity and sensitivity of the reagents are high.
  • 3 and 4 are graphs showing the results of the probe set 1. The results of probe set 2 were the same as those of probe set 1, and the figures are omitted.
  • Figure 4 shows the results of specimen 4 detection, the signal type is 2R2G, the EGFR gene is not amplified;
  • Figure 5 is the result of specimen 7 detection, the signal type is 6-12R2G, EGFR gene amplification.
  • the samples can be molecularly classified according to the test results, and used for clinical treatment plan formulation, drug selection and efficacy judgment according to the significance of the test indicators.
  • the detection can also be achieved by using one of the EGFR genes, respectively, and the specific results are omitted.
  • the detection signal can be better by using the combined probe. Theoretically, the longer the length of the probe, the brighter the fluorescence signal obtained during actual detection, but because more gene sequences may be involved, the complexity of the resulting signal is increased, and the difficulty of detection is also enhanced.
  • the total lengths of the BAC clones of the detection probes of Group 1 and Group 2 of the EGFR gene of the present invention are: 568 Kb and 380 Kb, respectively, which are nucleic acid mixtures comprising the EGFR gene and its both ends.

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Abstract

EGFR基因检测探针及其制备方法，制备方法包括选取BAC克隆为RP11-748O6、RP11-164G7、RP11-781C22、RP11-17J15中至少一种；或选取BAC克隆为CTD-2086F7、RP11-381G5、CTD-2343B15中的至少一种，对克隆分别提取质粒提取，得到质粒DNA，定量；用荧光素标记。提供包含EGFR检测探针的试剂盒。所述方法筛选得到的探针信号计数行准确、快速，且结果的重复性好；补充了临床中EGFR突变检测的不足。

Description

EGFR基因检测探针及其制备方法和试剂盒 技术领域
本发明属于生物技术，特别是涉及EGFR基因检测探针及其制备方法和试剂盒。
背景技术
肺癌是目前的研究热点，但检测热点主要集中在EGFR基因突变位点的检测，其在小分子TKI靶向治疗方面有潜在价值。但对于一些大分子单克隆抗体药物，如西妥昔单抗等，FISH检测EGFR拷贝数变化更具有参考价值。现在的临床研究也发现对于EGFR突变和EGFR扩增是一个部分重叠又独立的两个靶标，评价EGFR扩增能够有效预测患者的总生存，在临床用药方面也具有积极意义。
荧光原位杂交(Fluorescence in situ hybridization FISH)是20世纪80年代末期在原有的放射性原位杂交技术的基础上发展起来的一种非放射性原位杂交技术。目前这项技术已经广泛应用于动植物基因组结构研究、染色体精细结构变异分析、病毒感染分析、人类产前诊断、肿瘤遗传学和基因组进化研究待许多领域。FISH的基本原理是用已知的标记核酸为探针，按照碱基互补的原则，与待检材料中未知的单链核酸进行异性结合，形成可被检测的杂交双链核酸。由于DNA分子在染色体上是沿着染色体纵轴呈线性排列，因而可以探针直接与染色体进行杂交从而将特定的基因在染色体上定位。与传统的放射性标记原位杂交相比，荧光原位杂交具有快速、检测信号强、杂交特异性高和可以多重染色等特点，因此在分子细胞遗传学领域受到普遍关注。
杂交所用的探针大致可以分类三类：1)染色体特异重复序列探针，例如α卫星、卫星III类的探针，其杂交靶位常大于1Mb，不含散在重复序列，与靶位结合紧密，杂交信号强，易于检测；2)全染色体或染色体区域特异性探针，其由一条染色体或染色体上某一区段上极端不同的核苷酸片段所组成，可由克隆到噬菌体和质粒中的染色体特异大片段获得；3)特异性位置探针，由一个或几个克隆序列组成。
探针的荧光素标记可以采用直接和间接标记的方法。间接标记是采用生物素标记DNA探针，杂交之后用藕联有荧光素亲和素或者链霉亲和素进行检测，同时还可以利用亲和素-生物素-荧光素复合物，将荧光信号进行放大，从而可以检测500bp左右的片段。而直接标记法是将荧光素直接与探针核苷酸或磷酸戊糖骨架共价结合，或在缺口平移法标记探针时将荧光素核苷三磷酸掺入。直接标记法在检测时步骤简单，临床使用方便。
而目前对于EGFR基因FISH方法检测，还缺少特异性高的检测试剂盒。
发明内容
本发明的目的之一是提供一种EGFR基因检测探针及其制备方法，所制备的探针可用于检测EGFR基因状态，即检测EGFR基因的指拷贝数变化，具有很好的特异性。
实现上述目的的技术方案如下。
一种EGFR基因检测探针的制备方法，包括以下步骤：
(1)选取BAC克隆为RP11-748O6、RP11-164G7、RP11-781C22、RP11-17J15中至少一种，或选取BAC克隆为CTD-2086F7、RP11-381G5、CTD-2343B15中的至少一种；
(2)对克隆分别提取质粒，得到质粒DNA，定量；
(3)用荧光素标记质粒DNA，不同来源的质粒DNA所标记的荧光素相同，即得。
在其中一个实施例中，所述BAC克隆为RP11-748O6、RP11-164G7、RP11-781C22和RP11-17J15。
在其中一个实施例中，所述BAC克隆为CTD-2086F7、RP11-381G5和CTD-2343B15。
在其中一个实施例中，标记荧光素选择本领域已知的荧光染料，优选地，荧光素为Alexa
Figure PCTCN2016105716-appb-000001
FITC、Alexa
Figure PCTCN2016105716-appb-000002
Rhodamine、Texas Red、pacific
Figure PCTCN2016105716-appb-000003
DEAC。
在其中一个实施例中，基因探针的标记可以采用现有技术中的方法将相应荧光素标记至双链核酸上，所述方法包括但不限于：随机引物法、切口平移等，标记基因探针可以使用市售的缺口平移标记试剂盒和/或随机引物标记试剂盒，优选abbott和/或Roche公司的Nick Translation Kit。本发明步骤(3)优选采用随机引物法、切口平移法对质粒DNA进行荧光素标记。
本发明的另一目的是提供一种EGFR基因检测试剂盒。
实现该目的技术方案如下。
一种EGFR基因检测试剂盒，包括有上述EGFR基因检测探针。
在其中一个实施例中，包括有用于内控的7号染色体鉴别探针(CSP7)探针，该鉴别探针与EGFR基因检测探针标记的荧光素的颜色不相同。
在其中一个实施例中，还包括有用于封闭重复序列的COT Human DNA，和DAPI复染剂。
本发明具有以下有益效果：
1.本发明通过筛选到最优的EGFR基因检测探针及其组合，采用FISH(Fluorescence In-Situ Hybridization)方法对EGFR基因拷贝数检测，信号计数行准确、快速，且结果的重复性好；补充了临床中EGFR突变检测的不足，有利于筛选更多受益于靶向药物的患者，提高肺癌患者生存率和总生存期。
2.通过本发明所述的EGFR试剂盒，从基因水平了解EGFR状态改变，多种信号类型表现出实体组织的肿瘤细胞遗传多样性，可以实现在肿瘤生物学、细胞遗传学等领域的应用，有助综合评价各分子标志物，辅助肺癌临床靶向治疗用药及治疗方案选择。
附图说明
图1为是实施例1中检测探针序列的示意图。
图2是实施例1中人外周血培养细胞片FISH检测结果图。
图3为实施例3中EGFR基因检测基因扩增表现形式图。
图4为实施例4中肺癌组织样本FISH检测结果图，其中，检测信号类型为2R2G，EGFR基因未发生扩增。
图5为实施例4中肺癌组织样本FISH检测结果图，其中，检测信号类型为6-12R2G，EGFR基因扩增。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
实施例1EGFR基因检测探针的制备
本实施所述EGFR检测探针的制备方法，包括以下步骤：
(1)挑选包含目的基因EGFR及两端序列的克隆，如图1所示。
GSP EGFR包括两组，分别包括探针组1：第一探针、第二探针、第三探针和第四探针；探针组2：第一探针、第二探针和第三探针，具体如下表，其购买于Invitrogen RP11BAC及CTD BAC克隆库。以下分两组检测探针分别制备。
EGFR chr7:55,086,725-55,275,031,188,307bp
第一组
探针组1 BAC 插入片段起止位置
第一探针 RP11-748O6 chr7:54,711,888-54,884,289,172,402bp
第二探针 RP11-164G7 chr7:54,818,676-54,995,526,176,851bp
第三探针 RP11-781C22 chr7:54,987,970-55,164,028,176,059bp
第四探针 RP11-17J15 chr7:55,131,101-55,279,524,148,424bp
第二组
Figure PCTCN2016105716-appb-000004
(2)GSP EGFR基因检测探针制备：使用Qiagen公司的Plasmid Maxi Kit，按照说明书要求的操作方法对不同BAC克隆分别进行超低拷贝质粒DNA提取，通过测定260nm和280nm处的吸光度对质粒DNA定量；采用高压灭菌的超纯水稀释为200ng/ul,采用1.5ml的离心管分装，最后将分别得到的3种或4种质粒DNA混合，-20℃密封保存。
(3)通过切口平移方法对质粒DNA混合物进行荧光标记，每种探针标记的荧光素为Spectrum-Orange。采用abbott的Nick Translation Kit，按如下方案，严格避光条件下在冰上配制PCR反应体系。
Figure PCTCN2016105716-appb-000005
配完后震荡混匀，在16℃标记12小时，再80℃孵育10分钟灭活酶。取5ul使用2％琼脂糖凝胶做电泳，要求在50bp～500bp之间存在弥散的带。
对标记产物进行乙醇沉淀和浓缩，按如下方案在1.5ml离心管中依次加入 醋酸钠和无水乙醇，避光、冰上配制：
标记产物        45ul
醋酸钠(3mol/L)  5ul
无水乙醇        125ul
混匀后置于-70℃冰箱中至少2小时，4℃13000rpm离心30分钟，小心去上清，勿搅动沉淀，加入1ml的70％乙醇，4℃13000转/分钟离心15分钟，小心去上清，勿搅动沉淀，避光干燥。使用1ul纯化水溶解沉淀，获得GSP EGFR基因探针，避光、-20℃储存。
(4)GSP EGFR基因探针验证：分别使用两组探针，使用人类正常分裂中期淋巴细胞滴片进行待测样本进行两组探针验证(检测方法参考实施例3)。包含中期或间期染色体DNA,荧光原位杂交时，染色体DNA表现为形态上可识别的染色体或是细胞核。两组探针的结果相同，如图2所示：中期染色体的FISH杂交结果图。图中可以看见染色体7p11位置显示红色荧光信号，内控探针CSP7(7号染色体鉴别探针，可以标记出7号染色体，购自KREATECH-KBI20007)显示绿色荧光。
实施例2：EGFR基因检测试剂盒制备方法
EGFR基因检测试剂盒包括有EGFR杂交液和DAPI复染剂两个组分，其中EGFR杂交液包含实施例1所述的GSP EGFR基因探针(分别为两组检测探针，对应两种试剂盒)、CSP7探针(7号染色体鉴定探针)、用于杂交环境(促进杂交)的缓冲液组分、封闭重复序列的COT Human DNA等。DAPI复染剂主要用于杂交后的细胞复染，其中的DAPI会与DNA结合，使得细胞核显示出蓝色荧光，含有对苯二胺的复染剂可以保持荧光的稳定。
具体配方如下：
(1)杂交液配制
Figure PCTCN2016105716-appb-000006
(2)DAPI复染剂配制
10mg的对苯二胺溶于1ml的PBS中，调节pH为9.0，加入9ml甘油，反复震荡混匀，-20℃储存。取2.5μl的DAPI溶液(0.1mg/ml)溶于1ml抗褪色液中，避光条件下反复震荡混匀，-20℃避光密闭保存。
(3)成品组装
Figure PCTCN2016105716-appb-000007
实施例3：EGFR基因检测试剂盒的检测方法
1、玻片预处理
1.1玻片放入65±5℃恒温箱中烤片过夜；
1.2取出玻片，将其放入二甲苯中室温脱蜡15分钟；
1.3取出玻片，再将其放入另一缸二甲苯中室温继续脱蜡15分钟；
1.4取出玻片，再将其放入无水乙醇中室温10分钟，去除残留二甲苯；
1.5取出玻片，再将其放入100％、90％、70％梯度乙醇室温复水各3分钟；
1.6取出玻片，再将其放入纯化水中室温洗涤3分钟，用无绒纸巾吸取多余水分；
1.7取出玻片，再将其放入纯化水中100±5℃煮片25分钟(切片水平放置于容器中，样本面朝上)；
1.8取出玻片，室温晾干；
1.9将玻片正面朝上放在架子上，在样本区域滴加适量的胃蛋白酶反应液，消化5～15分钟；
1.10将多余液体甩去，将其放入室温2×SSC中5分钟；
1.11取出玻片，再将其放入另一缸室温2×SSC中5分钟；
1.12取出玻片，再将其依次放入室温70％，90％，100％梯度乙醇脱水各3分钟；
1.13取出玻片，室温晾干。
胃蛋白酶的反应时间需要通过预试验进行确定。可以使用同批制备的样本片按所述方法进行预试验，通常以5分钟为间隔时间。例如，分别测试消化时间为5分钟、10分钟和15分钟，完成“玻片预处理”后，可以在明场下，使用10×或20×物镜观察组织消化状态；或者直接进行DAPI复染，进行消化状态判断。
2、样品和探针同时变性(避光操作)
2.1从-20±5℃冰箱中取出实施例2所述检测试剂盒中的杂交液，震荡混匀，瞬时离心；
2.2加10μl的杂交液到杂交区域，迅速盖上18×18mm盖玻片，轻压使杂交 液均匀分布，避免产生气泡；
2.3用橡皮胶沿盖玻片边缘封片，完全覆盖盖玻片和载玻片接触的部位；
2.4将玻片放入杂交仪中，湿润原位杂交仪湿度条，插入湿条，盖上杂交仪上盖，设置“Denat&Hyb”程序，变性85℃ 5分钟，杂交37℃ 10～18小时。(若无杂交仪，可使用替代仪器，如恒温热台进行变性，电热烘箱/或水浴锅进行杂交，需注意温度准确及保持杂交湿度)。
3、杂交后洗涤及复染(避光操作)
3.1洗涤前30分钟，将配制好的洗液I,洗液II，放入37±1℃的水浴中，测量以确保温度合适；
3.2关闭杂交仪电源，将玻片取出，轻轻撕去橡皮胶，移去盖玻片(若盖玻片难以去除，可以将其放入洗液I中微微摇晃，以利于其脱落；
3.3玻片放入37±1℃洗液I(2×SSC)中10分钟；
3.4取出玻片，再将其放入37±1℃洗液II(0.1％NP-40/2×SSC)中5分钟；
3.5取出玻片，室温70％乙醇中3分钟；
3.6取出玻片，暗处自然干燥玻片；
3.7室温，滴加10μl DAPI复染剂到22×22mm的盖玻片,载玻片目标区域朝下，轻放于盖玻片上，轻压，避免产生气泡，在暗处存放，待观察。
上述所列举试剂均在圆形染色缸中配制(每种试剂体积均为40ml)，每个染色缸最多可放入5片切片。非室温溶液，在操作开始前需提前预热反应试剂至指定温度。在洗涤过程中，可间隔2～3分钟轻轻晃动染色缸，提高洗涤效果。
4、结果分析
相关荧光和DAPI需用合适的滤块观察。其中，CSP7探针显示绿色信号； GSP EGFR探针为红色信号。
4.1使用合适的滤镜，在40×物镜下寻找，在100×物镜下计数；
4.2调整合适的焦距，对信号和背景有明确的概念；信号点因位于细胞内；当细胞外存在荧光信号点时,要注意与细胞内信号点区分，最好能避开该区域进行计数；
4.3扫视几个肿瘤细胞区域,选择至少4个有很好核分界的区域,要求细胞核边界完整，DAPI染色均匀、核无重叠，CSP7探针(绿色信号点)信号清晰；
4.4从选择区域的左上角开始分析，从左到右扫视，观察多个视野；
4.5组织计数的要求：
a.只计数肿瘤组织(在FISH检测前，使用HE染色片进行对照观察)
b.避免在坏死区域及核边界不清的区域计数
c.需要主观辨别的核不计数
d.跳过信号弱及没有特定信号或高背景的核计数
4.6转至100×物镜，调整焦距，在核的不同层次找到所有信号点；
4.7在每个核内计数信号点；调焦找到每个核内的所有信号点，计数一个区域内的两种信号，只计数每种颜色有1个或更多FISH信号的，没有信号或只有一种颜色信号的核不计数；记录观察到的细胞总数(信号正常及异常)；
4.8计数方法
在5个清晰的肿瘤区域，共计数50个肿瘤细胞核内GSP EGFR(红色)和CSP 7(绿色)信号，分别计数单个细胞核内GSP EGFR和CSP 7的信号数。计数时，要特别注意信号断裂、成对或是成簇情况。当两个或三 个信号由线状相连或是相邻(间距小于信号点直径)时作为一个信号。记录最大信号数目为15，当＞15时，记录为15。
5、试验结果的判定
统计GSP EGFR信号≥4个的细胞占统计总细胞的百分比，再计算所有细胞GSP EGFR信号总数与CSP 7信号数的比率。
当如下情况时：
a.≥40％的细胞出现≥4个EGFR信号；
b.GSP EGFR/CSP7(红色/绿色)≥2，同时，用于计数的核中CSP7信号≥2；
c.≥10％的细胞出现≥4个EGFR信号簇；
d..≥10％的细胞出现＞15个EGFR信号；
若满足情况a、或b、或c、或d时判定为FISH阳性(EGFR基因扩增)；
不满足上述情况判定为FISH阴性(EGFR基因无扩增)。
研究表明，在非小细胞肺癌样本中EGFR基因扩增表现形式多样，基于FISH检测，常表现为6种信号形式：在约60％的核中EGFR信号表现为相对松散、超过20个拷贝的信号类型，扩增子表现为均染区(图3a)；在10％～15％核中EGFR信号表现为4-10拷贝的基因信号(图3b)；在15％-20％核中扩增子包含CSP7序列，表现为共扩增基因簇(图3c)；约5％核中出现非典型的相对内对照细胞内信号大且明亮的基因信号簇(图3d)；约1％核内出现双微体(图3e)；5％核内出现染色体非整倍情况，表现为≥10％核内出现≥15EGFR信号(图3f)。
实施例4：EGFR基因检测试剂盒临床使用评价
使用实施例1所述两组检测探针，实施例2所述检测试剂盒对20份临床样本(其经过病理检测确诊，具体见下表)，进行检测。根据实施例3的检测方法重复检测3次，结果相符，检测结果的重复性好；两种探针组合的检测一致性佳。与市售商品化试剂比较，检测结果完全一致，试剂的特异性和灵敏度高。图3和图4为探针组1的检测结果图。探针组2的结果与探针组1的结果相同，图省略。图4为标本4检测结果，信号类型为2R2G，EGFR基因未发生扩增；图5为标本7检测结果，信号类型为6-12R2G，EGFR基因扩增。
Figure PCTCN2016105716-appb-000008
Figure PCTCN2016105716-appb-000009
从检测结果知，在对这些样本进行分子标志物检测后，可以据检测结果对样本进行分子分型，依据检测指标的意义，用于临床治疗方案制定、用药选择和疗效判断。
本发明中，分别各使用EGFR基因中的一种探针也能实现相应的检测，具体结果省略，但相对探针组合使用而言，组合探针的使用，检测信号会更好。理论上探针长度越长，实际检测时获得的荧光信号亮度越明亮，但因为可能涉及到更多基因序列，所得到的信号复杂性可能性增多，对检测实现的难度也增强。本发明所述针对EGFR基因的组1和组2的检测探针的BAC克隆总长度分别为：568Kb和380Kb,均为包含EGFR基因及其两端序列的核酸混合物。
发明人在对本发明所述探针验证中发现，较长的检测探针确实获得更强的荧光信号，并且在对临床样本的检测验证中也获得了相同的结果。因此，在荧光探针的设计中，可以通过适当延长荧光探针长度增加信号亮度，但具体如何组合使用，存在的一定的技术困难，要实现很好的检测结果，除了设计中的经验之外，还需通过临床样本验证评估信号类型差异，需要发明人付 出大量艰辛的工作，寻求合适的探针组合。
所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (8)

  1. 一种EGFR基因检测探针的制备方法，其特征在于，包括以下步骤：
    (1)选取BAC克隆为RP11-748O6、RP11-164G7、RP11-781C22、RP11-17J15中至少一种，或选取BAC克隆为CTD-2086F7、RP11-381G5、CTD-2343B15中的至少一种；
    (2)对BAC克隆分别提取质粒，得到质粒DNA，定量；
    (3)用荧光素标记质粒DNA，不同来源的质粒DNA所标记的荧光素相同，即得。
  2. 根据权利要求1所述EGFR基因检测探针的制备方法，其特征在于，所述BAC克隆为RP11-748O6、RP11-164G7、RP11-781C22和RP11-17J15；或所述BAC克隆为CTD-2086F7、RP11-381G5和CTD-2343B15。
  3. 根据权利要求1所述EGFR基因检测探针的制备方法，其特征在于，所述荧光素为Alexa
    Figure PCTCN2016105716-appb-100001
    FITC、Alexa
    Figure PCTCN2016105716-appb-100002
    Rhodamine、Texas Red、pacific
    Figure PCTCN2016105716-appb-100003
    DEAC。
  4. 根据权利要求1-3任一项所述EGFR基因检测探针的制备方法，其特征在于，步骤(3)采用随机引物法或切口平移法对质粒DNA进行荧光素标记。
  5. 根据权利要求1-4任一项所述的制备方法得到的EGFR基因检测探针。
  6. 一种EGFR基因检测试剂盒，其特征在于，包括有权利要求5所述的EGFR基因检测探针。
  7. 根据权利要求6所述EGFR基因检测试剂盒，其特征在于，还包括有用于内控的7号染色体鉴别探针，该鉴别探针与EGFR基因检测探针标记的荧光素的颜色不相同。
  8. 根据权利要求6或7所述EGFR基因检测试剂盒，其特征在于，还 包括有用于封闭重复序列的COT Human DNA，和DAPI复染剂。
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