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WO2017114003A1 - Alk基因和eml4基因检测探针及其制备方法和试剂盒 - Google Patents

Alk基因和eml4基因检测探针及其制备方法和试剂盒 Download PDF

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WO2017114003A1
WO2017114003A1 PCT/CN2016/105703 CN2016105703W WO2017114003A1 WO 2017114003 A1 WO2017114003 A1 WO 2017114003A1 CN 2016105703 W CN2016105703 W CN 2016105703W WO 2017114003 A1 WO2017114003 A1 WO 2017114003A1
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gene
alk
eml4
ctd
alk gene
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何瑰
陈绍宇
欧焕金
张会清
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Guangzhou LBP Medicine Science and Technology Co Ltd
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Definitions

  • the present invention belongs to the field of biotechnology, and particularly relates to an ALK gene and EML4 gene detecting probe, a preparation method thereof and a kit.
  • lung cancer ranked first among the national incidence of malignant tumors (35.23/100,000, 19.59%) and mortality (27.93/100,000, 24.87%).
  • the occurrence of lung cancer is considered to be a multi-step process that is closely related to the amplification/activation of protooncogenes and/or the inactivation of tumor suppressor genes, and provides targets for patients as more and more cancer-driven factors are discovered.
  • the opportunity to treat The study found that 64% of patients with lung adenocarcinoma detected cancer-driven factors, of which 8% were ALK rearrangements.
  • ALK gene fusion is not a favorable prognostic factor for advanced NSCLC, but crizotinib improves survival in ALK-positive patients.
  • EML4-ALK echinoderma microtubule-associated protein-like 4-anaplastic lymphoma kinase
  • fusion gene mutation is the main type, accounting for about 5% of all NSCLC.
  • Other genes that are fused to the ALK gene in lung cancer include TFG, KIF5B, and the like.
  • the EML4-ALK fusion gene can be found in a variety of tumors, such as anaplastic large cell lymphoma, inflammatory myofibroblastoma, neuroblastoma, and NSCLC, which are caused by short arm insertion of chromosome 2.
  • the ALK fusion gene forms an intricate signal transduction network, which affects cell proliferation, differentiation and apoptosis through the activation and transmission of downstream substrate molecules and the mutual crossing and overlapping of transduction pathways.
  • the EML4-ALK fusion gene binds the kinase regions of two EML4-ALK molecules through the extracellular helical domain of the fusion partner to form a stable dimer, which activates downstream MAPK, PI3K/AKT, JAK/STAT3 by autophosphorylation. Wait for the pathway, causing the cells to metastasize.
  • ALK gene rearrangement was first discovered and named in anaplastic lymphoma, and was reported in lung cancer in 2007. Now, as a member of the tyrosine kinase signaling pathway, ALK gene abnormalities may be found in more cancerous species, and the indications for targeted drugs will continue to expand.
  • 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 ALK gene detecting probe and a preparation method thereof, and the prepared probe can be used for detecting ALK gene rearrangement and identification, and has good specificity.
  • a method for preparing an ALK gene detection probe comprising the steps of:
  • the BAC clones targeting the ALK gene were selected as at least one of RP11-62B19, RP11-100C1 and CTD-2245E6 as a set of probes, and the BAC clones were selected as CTD-2544I11, RP11-684O3 and RP11-134O13. At least one as another set of probes;
  • the color of the fluorescein of the same group of probes is the same, the color of the fluorescein of different sets of probes is different, that is, the BAC clones are RP11-62B19, RP11-100C1 and CTD-2245E6.
  • the probe is different from the color of the fluorescein derived from the probes of the BAC clones CTD-2544I11, RP11-684O3 and RP11-134O13.
  • Another aspect of the present invention provides an ALK gene and EML4 gene detecting probe and a preparation method thereof, and the prepared probe can be used for detecting the ALK gene and the EML4 gene state, that is, for EML4/ALK gene fusion identification, which is excellent. Specificity.
  • a method for preparing an ALK gene and an EML4 gene detection probe comprising the steps of:
  • the BAC clones targeting the ALK gene were selected as at least one of RP11-62B19, RP11-100C1 and CTD-2245E6 as a set of probes, and the BAC clones were selected as CTD-2544I11, RP11-684O3 and RP11-134O13. At least one of the other set of probes; and the BAC clone selected for the EML4 gene is at least one of CTD-2358L8 and CTD-2547J15;
  • the color of the fluorescein labeled with the ALK gene and the detection probe for the EML4 gene is different, and for the ALK gene, the color of the fluorescein of the same group of probes is the same, different groups
  • the color of the fluorescein of the needle is different, that is, the probe derived from BAC clones as RP11-62B19, RP11-100C1 and CTD-2245E6 and the probe derived from BAC clones as CTD-2544I11, RP11-684O3 and RP11-134O13.
  • the color of fluorescein is not the same, that is.
  • the ALK rearrangement probe comprises two groups, the first group of BAC clones being RP11-62B19, RP11-100C1 and CTD-2245E6, respectively; the second group of BAC clones being CTD-2544I11, RP11- 684O3 and RP11-134O13.
  • the EML4 probe comprises a set and the third set of BAC clones are CTD-2358L8 and CTD-2547J15.
  • 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 used with a commercially available nick translation labeling kit and a random primer labeling kit, preferably abbott and Roche's Nick Translation Kit.
  • the plasmid DNA is preferably subjected to fluorescein labeling by a random primer method or a nick translation method.
  • the temperature of the label is between 15 ° C and 18 ° C and the time of labeling is between 8 and 12 hours.
  • Another object of the present invention is to provide an ALK gene and EML4 gene detecting kit.
  • a kit for detecting ALK gene and EML4 gene comprising the above ALK gene and EML4 gene detecting probe.
  • the present invention can detect unknown rearrangement associated with ALK or directly identify EML4/ALK rearrangement by screening for optimal EML4/ALK gene rearrangement detection probes and combinations thereof.
  • the signal counting is accurate and rapid, and the result is reproducible; supplementing the shortage of detection reagents in the clinic depends on the import, which is beneficial to the subsequent detection probe. It is used in clinical research for molecular typing and targeted therapy of lung cancer.
  • Fig. 1A is a schematic diagram showing the sequence of the ALK gene detecting probe in Example 1.
  • Fig. 1B is a schematic diagram showing the probe sequence of the EML4 gene detection in Example 1.
  • Fig. 2A is a graph showing the results of FISH detection of the human peripheral blood culture cell sheet ALK gene detecting probe in Example 1.
  • Fig. 2A is a graph showing the results of FISH detection of the human peripheral blood culture cell sheet ALK gene and EML4 gene detection probe in Example 1.
  • Fig. 3A is a diagram showing the results of FISH detection of the ALK gene of the lung cancer tissue sample in Example 4, wherein the detection signal type is 2F (RG adjacent or coincident), and the ALK gene is not rearranged.
  • FIG. 3B is a diagram showing the results of FISH detection of the ALK gene and the EML4 gene of the lung cancer tissue sample in Example 4, wherein the detection signal type is 2F2A, the ALK gene is not rearranged, and no EML4/ALK rearrangement occurs.
  • FIG. 4A is a diagram showing the results of FISH detection of the ALK gene of the lung cancer tissue sample in Example 4, wherein the detection signal type is 1F1G1R, and the F signal is broken compared with the normal signal type 2F, and the detection result is: ALK gene rearrangement.
  • FIG. 4B is a diagram showing the results of FISH detection of the ALK gene and the EML4 gene in the lung cancer tissue sample of Example 4, wherein the detection signal type is 1F1A1G1RA, and the F signal is broken and the RA signal fusion occurs simultaneously with the normal signal type 2F2A.
  • ALK gene is broken and the rearrangement type is EML4/ALK.
  • GSP ALK includes two sets of probes:
  • the first set of probes included a first probe, a second probe, and a third probe, as shown in the following table, which was purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • the second set of probes included a fourth probe, a fifth probe, and a sixth probe, as specifically listed below, which were purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • Probe set 1 (red) BAC Insert start and end position First probe RP11-62B19 Chr2:29354267..29528536 Second probe RP11-100C1 Chr2:29210476..29383355 Third probe CTD-2245E6 Chr2:29066898..29207778
  • Probe set 2 (green) BAC Insert start and end position Fourth probe CTD-2544I11 Chr2:29923580..30121092 Fifth probe RP11-684O3 Chr2:30116506..30315853 Sixth probe RP11-134O13 Chr2:30282641..30436938
  • the preparation method of the EML4 detection probe of the present embodiment comprises the following steps:
  • GSP EML4 includes a set of probes including a first probe and a second probe, as shown in the following table, which was purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • the labeled product was subjected to ethanol precipitation and concentration, and sodium acetate and absolute ethanol were sequentially added to a 1.5 ml centrifuge tube in the following manner, and protected from light and ice:
  • GSP ALK and GSP EML4 gene probe validation hybridization solution prepared using ALK probe set 1 + ALK probe set 2+ EML4 probe set 3, respectively, and ALK probe set 1 + ALK probe set 2 preparation
  • the hybridization solution is subjected to probe verification on human normal division metaphase lymphocyte droplets (detection method is referred to in Example 3).
  • the cultured cells contain metaphase or interphase chromosomal DNA. When fluorescent in situ hybridization, the chromosomal DNA appears as a morphologically recognizable chromosome or nucleus.
  • FISH hybridization of metaphase chromosomes as shown in Fig. 2A (experimental results applied to probe set 1 + probe set 2) and Fig.
  • FIG. 2A shows the upper metaphase chromosome of human peripheral blood cultured cells.
  • the figure shows that the ALK gene (2p23) segment of chromosome 2 shows a red-green fusion signal (yellow), the other segments have no hybridization signal point, and the ALK probe group 1 and probe Group 2 has excellent specificity.
  • Fig. 2B shows the upper metaphase chromosome of human peripheral blood culture cells. The red-green fusion signal of 2p23 segment (yellow), the cyan signal of 2p21 segment, the hybridization signal point of other segments, ALK probe group 1, ALK probe group are shown in the figure. 2 and EML4 probe set 3 have superior specificity.
  • Example 2 Kit for ALK gene detection, ALK gene and EML4 gene detection
  • This embodiment provides two kits, namely an ALK gene detection kit, and an ALK gene and EML4 gene detection kit.
  • the ALK gene rearrangement detection kit includes two components of a hybridization solution and a DAPI counterstain, wherein the hybridization solution comprises the GSP ALK two-set probe (two-color) described in Example 1, and is used in a hybrid environment (promoting hybridization). Buffer components, closed repeats of COT Human DNA, 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 ALK gene and EML4 gene detection kit includes two components of ALK/EML4 hybridization solution and DAPI counterstaining agent, wherein the ALK and EML4 hybridization solution comprises the set of GSP EML4 gene probes described in Example 1 and two sets of GSP ALK. A combination of gene probes.
  • the ALK gene has two sets of detection probes, and the EML4 gene has a set of detection probes.
  • the ALK gene and the EML4 gene detection kit are: ALK (group 1+group 2)+EML4 (group 3) Combinations, buffer components for hybrid environment (promoting hybridization), CDM Human DNA such as closed repeats, DAPI counterstains are mainly used for counterstaining of cells after hybridization, in which DAPI binds to DNA, allowing the nucleus to show Blue fluorescence, a counterstain containing p-phenylenediamine, maintains fluorescence stability.
  • the relevant fluorescence and DAPI need to be observed with a suitable filter block.
  • Each negative control panel randomly counts the complete 100 cells, counts the number and percentage of abnormal signal cells in each sample, the average and standard deviation of the statistical percentage, and the negative threshold is set to the mean + 3 standard deviation .
  • the two detection kits described in Example 2 one is a combination of (ALK (Group 1) + ALK (Group 2) + EML4 (Group 3) (hereinafter referred to as EML4/ALK rearrangement test kit), and another kit for combination of ALK (group 1) + ALK (group 2) (hereinafter referred to as ALK rearrangement detection kit), for 20 parts
  • Clinical samples which were confirmed by pathological examination, the specimens were formalin-fixed paraffin-embedded tissue samples, see the table below), and were tested separately. The experiments of the two kits were repeated three times, and the test results were repeated. The same, the detection consistency is good.
  • FIG. 3A is a diagram of the FISH detection result of the lung tissue sample of the ALK rearrangement detection kit, wherein the detection signal type For 2F (RG adjacent or coincident), the ALK gene is not rearranged.
  • FIG. 3A is a diagram of the FISH detection result of the lung tissue sample of the ALK rearrangement detection kit, wherein the detection signal type For 2F (RG adjacent or coincident), the ALK gene is not rearranged.
  • FIG. 3B is a diagram showing the results of FISH detection of lung cancer tissue samples by the EML4/ALK rearrangement detection kit, wherein the detection signal type is 2F2A, the ALK gene is not rearranged, and no EML4/ALK rearrangement occurs.
  • FIG. 3B is a diagram showing the results of FISH detection of lung cancer tissue samples in the examples, wherein the detection signal type is 2F2A, the ALK gene is not rearranged, and no EML4/ALK rearrangement occurs.
  • FIG. 4A is a diagram showing the results of FISH detection of lung cancer tissue samples by ALK rearrangement detection kit, wherein the detection signal type is 1F1G1R, and the F signal is broken compared with the normal signal type 2F, and the detection result is: ALK gene rearrangement.
  • Figure 4B is a diagram showing the results of FISH detection of lung cancer tissue samples by EML4/ALK rearrangement detection kit.
  • the detection signal type is 1F1A1G1RA. Compared with the normal signal type 2F2A, F signal rupture occurs and RA signal fusion occurs at the same time.
  • the detection result is : ALK gene is broken and the rearrangement type is EML4/ALK.
  • the detection kits of the EML4 gene and the ALK gene gene rearrangement and fusion type detection are performed using three sets of probes, respectively.
  • the length of the probe group 3 of the EML4 gene, the probe set 1 of the ALK gene, and the probe set of the probe set 2 of the ALK gene of the present invention are 261 Kb, 462 Kb, and 513 Kb, respectively.
  • the ALK rearrangement partner has many genes, and EML4/ALK rearrangement is one of them.
  • the ALK gene rearrangement detection probe (including the ALK two-group probe) can detect ALK rearrangement, and breakage means that rearrangement occurs, but the rearrangement type cannot be judged.
  • the EML4/ALK rearrangement detection probe (including the ALK two-set probe and the EML4 set of probes) can detect ALK rearrangement and simultaneously determine whether EML4/ALK rearrangement has occurred.
  • the samples can be molecularly classified according to the detection results, and used for clinical treatment plan formulation, drug selection and efficacy judgment according to the significance of the detection indicators.

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Abstract

本发明公开了ALK基因检测探针，以及ALK基因和EML4基因检测探针及其制备方法，该方法包括以下步骤：选取针对ALK基因的BAC克隆为RP11-62B19、RP11-100C1和CTD-2245E6中至少一种，和选取BAC克隆为CTD-2544I11、RP11-684O3和RP11-134O13中的至少一种；以及选取针对EML4基因的BAC克隆为CTD-2358L8和CTD-2547J15中至少一种；得到质粒DNA；标记。本发明还公开了包含有ALK基因和EML4基因检测探针的试剂盒。

Description

ALK基因和EML4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ALK基因和EML4基因检测探针及其制备方法和试剂盒。
背景技术
2014年，肿瘤登记年报提示肺癌在全国恶性肿瘤的发病率(35.23/10万，19.59％)和死亡率(27.93/10万，24.87％)中居第一位。肺癌的发生被认为是一个多步骤过程，与原癌基因的扩增/激活和/或肿瘤抑癌基因的失活密切相关，随着越来越多癌症驱动因子的发现，为患者提供了靶向治疗的机会。研究发现，64％的肺腺癌患者检测出癌症驱动因子，其中，8％为ALK重排。ALK基因融合不是晚期NSCLC的有利预后因素，但crizotinib可改善ALK阳性患者的生存。既往研究显示，EML4-ALK基因融合变异率为2％～7％。2011年美国临床肿瘤学会(ASCO)年会报道的一项研究显示，经EML4-ALK抑制剂crizotinib治疗的ALK阳性者的1年、2年生存率分别为74％和54％，且接受crizotinib二、三线治疗ALK阳性者的生存率显著高于ALK阳性对照组和ALK阴性对照组。肺癌中ALK变异主要为ALK基因发生重排与其他基因融合。EML4-ALK(棘皮动物微管结合蛋白样4-间变性淋巴瘤激酶)融合基因变异是其主要类型，约占所有NSCLC的5％左右。肺癌中与ALK基因融合的其他基因还包括TFG、KIF5B等。EML4-ALK融合基因可见于多种肿瘤，例如间变性大细胞淋巴瘤、炎性成肌纤维细胞瘤、成神经细胞瘤和NSCLC等，它是由第2号染色体短臂插入引起。ALK融合基因通过下游底物分子的激活、传递，各转导途径的相互交叉、重合，形成了一个错综复杂的信号转导网络，影响细胞增殖、分化和凋亡。EML4-ALK融合基因通过融合伴侣的胞外螺旋结构域，使两个EML4-ALK分子的激酶区相互结合，形成稳定的二聚体，通过自身磷酸化活化下游MAPK、PI3K/AKT、JAK/STAT3等通路，从而引起细胞向恶性转化。
ALK基因重排最早在间变性淋巴瘤中被发现和命名，2007年在肺癌中发 现，作为酪氨酸激酶信号通路的一员，ALK基因异常可能在更多的癌种中被发现，并且其靶向药物的适应症也将不断扩展。
因此，研究用于该基因的检测试剂盒，并用于临床样本检测，对于筛选靶向药物受益人群极有益处。
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ALK/EML4基因FISH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ALK基因检测探针及其制备方法，所制备的探针可用于检测ALK基因重排鉴定，具有很好的特异性。
实现上述目的的技术方案如下。
一种ALK基因检测探针的制备方法，包括以下步骤：
(1)选取针对ALK基因的BAC克隆为RP11-62B19、RP11-100C1和CTD-2245E6中至少一种作为一组探针，和选取BAC克隆为CTD-2544I11、RP11-684O3和RP11-134O13中的至少一种作为另一组探针；
(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，同组探针的荧光素的颜色相同，不同组探针的荧光素的颜色不相同，即来源于BAC克隆为RP11-62B19、RP11-100C1和CTD-2245E6的探针与来源于BAC克隆为CTD-2544I11、RP11-684O3和RP11-134O13的探针的荧光素的颜色不相同，即得。
本发明的另一是提供一种ALK基因和EML4基因检测探针及其制备方法，所制备的探针可用于检测ALK基因和EML4基因状态，即用于EML4/ALK基因融合鉴定，具有很好的特异性。
一种ALK基因和EML4基因检测探针的制备方法，包括以下步骤：
(1)选取针对ALK基因的BAC克隆为RP11-62B19、RP11-100C1和CTD-2245E6中至少一种作为一组探针，和选取BAC克隆为CTD-2544I11、RP11-684O3和RP11-134O13中的至少一种作为另一组探针；以及选取针对EML4基因的BAC克隆为CTD-2358L8和CTD-2547J15中至少一种；
(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，针对ALK基因和针对EML4基因的检测探针标记的荧光素的颜色不相同，且针对ALK基因中，同组探针的荧光素的颜色相同，不同组探针的荧光素的颜色不相同，即来源于BAC克隆为RP11-62B19、RP11-100C1和CTD-2245E6的探针与来源于BAC克隆为CTD-2544I11、RP11-684O3和RP11-134O13的探针的荧光素的颜色不相同，即得。
在其中一个实施例中，所述ALK重排探针包括两组，分别为第一组BAC克隆为RP11-62B19、RP11-100C1和CTD-2245E6；第二组BAC克隆为CTD-2544I11、RP11-684O3和RP11-134O13。
在其中一个实施例中，所述EML4探针包括一组，第三组BAC克隆为CTD-2358L8和CTD-2547J15。
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 PCTCN2016105703-appb-000001
FITC、Alexa
Figure PCTCN2016105703-appb-000002
Rhodamine、Texas Red、pacific
Figure PCTCN2016105703-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随机引物标记试剂盒，优选abbott和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。
在其中一个实施例中，所述标记的温度为15℃-18℃，标记的时间为8-12小时。
本发明的另一目的是提供一种ALK基因和EML4基因检测试剂盒。
实现该目的技术方案如下。
一种ALK基因和EML4基因检测试剂盒，包括有上述ALK基因和EML4基因检测探针。
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EML4/ALK基因重排检测探针及其组合，既可以检测未知的与ALK相关重排，也可以直接鉴别EML4/ALK重排。
(2)采用本发明所述的方法对ALK基因重排进行检测，信号计数行准确、快速，且结果的重复性好；补充了临床中检测试剂依赖进口的不足，有利于后续该检测探针应用于临床研究，进行肺癌分子分型，靶向治疗等方面。
附图说明
图1A为是实施例1中ALK基因检测探针序列的示意图。
图1B为是实施例1中EML4基因检测探针序列的示意图。
图2A为实施例1中人外周血培养细胞片ALK基因检测探针FISH检测结果图。
图2A为实施例1中人外周血培养细胞片ALK基因和EML4基因检测探针FISH检测结果图。
图3A为实施例4中肺癌组织样本ALK基因FISH检测结果图，其中，检测信号类型为2F(RG相邻或重合)，ALK基因未重排。
图3B为实施例4中肺癌组织样本ALK基因和EML4基因FISH检测结果图，其中，检测信号类型为2F2A，ALK基因未重排，未发生EML4/ALK重排。
图4A为实施例4中肺癌组织样本ALK基因FISH检测结果图，其中，检测信号类型为1F1G1R，对比正常信号类型2F，发生了F信号断裂，检测结果为：ALK基因重排。
图4B为实施例4中肺癌组织样本ALK基因和EML4基因FISH检测结果图，，其中，检测信号类型为1F1A1G1RA，对比正常信号类型2F2A，发生了F信号断裂，并同时发生RA信号融合，检测结果为：ALK基因断裂，重排类型为EML4/ALK。
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1  ALK基因和EML4基因检测探针的制备
本实施所述ALK检测探针的制备方法，包括以下步骤：
(1)挑选包含目的基因ALK及两端序列的克隆，如图1A所示。GSP ALK包括两组探针：
第一组探针包括第一探针、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。
第二组探针包括第四探针、第五探针和第六探针，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。
探针组1—标记红色R，BAC总长度：462Kb。
探针组2—标记绿色G，BAC总长度：513Kb。
探针组1(红) BAC 插入片段起止位置
第一探针 RP11-62B19 chr2:29354267..29528536
第二探针 RP11-100C1 chr2:29210476..29383355
第三探针 CTD-2245E6 chr2:29066898..29207778
探针组2(绿) BAC 插入片段起止位置
第四探针 CTD-2544I11 chr2:29923580..30121092
第五探针 RP11-684O3 chr2:30116506..30315853
第六探针 RP11-134O13 chr2:30282641..30436938
本实施所述EML4检测探针的制备方法，包括以下步骤：
挑选包含目的基因EML4近ALK端序列的克隆，如图2B所示。GSP EML4包括一组探针，该组探针有包括第一探针和第二探针，具体如下表，其购买于Invitrogen RP11BAC及CTD BAC克隆库。
探针组3-标记青色A。
BAC总长度：261Kb
探针组3(青色) BAC 插入片段起止位置
第一探针 CTD-2358L8 chr2:42305395..42410427
第二探针 CTD-2547J15 chr2:42149101..42340641
(2)GSP ALK和GSP EML4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，最后将得到的对应ALK基因或EML4基因的3种或2种相应组合的质粒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，针对GSP ALK基因的组2探针标记的荧光素为green-dUTP，组3探针标记的荧光素Aqua-dUTP，针对GSP EML4基因每种探针标记的荧光素为red-dUTP。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 PCTCN2016105703-appb-000004
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，分别在50-500bp左右存在弥散的条带。
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 ALK和GSP EML4基因探针，避光、-20℃储存。
(4)GSP ALK和GSP EML4基因探针验证：分别使用ALK探针组1+ALK探针组2+EML4探针组3制备的杂交液，以及ALK探针组1+ALK探针组2制备的杂交液，对人类正常分裂中期淋巴细胞滴片进行探针验证(检测方法参考实施例3)。培养细胞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。如图2A(应用于探针组1+探针组2的实验结果)和图2B(应用于探针组1+探针组2+探针组3的实验结果)所示中期染色体的FISH杂交结果图。图2A示人外周血培养细胞片上中期染色体，图中可见2号染色体ALK基因(2p23)区段显示红绿融合信号(黄色)，其它区段未见杂交信号点，ALK探针组1和探针组2的特异性优。图2B示人外周血培养细胞片上中期染色体，图中可见2p23区段红绿融合信号(黄色)，2p21区段青色信号，其它区段未见杂交信号点，ALK探针组1、ALK探针组2和EML4探针组3的特异性优。
实施例2：ALK基因检测、ALK基因和EML4基因检测的试剂盒
本实施例提供2种试剂盒，分别为ALK基因检测试剂盒，以及ALK基因和EML4基因检测试剂盒。
ALK基因重排检测试剂盒包括有杂交液和DAPI复染剂两个组分，其中杂交液包含实施例1所述的GSP ALK两组探针(双色)、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：
杂交液配制
Figure PCTCN2016105703-appb-000005
ALK基因和EML4基因检测试剂盒包括有ALK/EML4杂交液和DAPI复染剂两个组分，其中ALK和EML4杂交液包含实施例1所述的一组GSP EML4基因探针和两组GSP ALK基因探针的组合。ALK基因有两组检测探针，EML4基因有一组检测探针，本实施例中，所述ALK基因和EML4基因检测试剂盒，为：ALK(组1+组2)+EML4(组3)的组合、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 PCTCN2016105703-appb-000006
Figure PCTCN2016105703-appb-000007
(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
组分名称 规格/10test 数量
杂交液 100μl/管 1管
DAPI复染剂 100μl/管 1管
说明书   1份
实施例3：ALK基因和EML4基因检测试剂盒的检测方法
1、玻片预处理
1.玻片放入65±5℃恒温箱中烤片过夜；
2.取出玻片，将其放入室温二甲苯(或环保脱蜡剂)中10分钟；
3.取出玻片，再将其放入另一缸室温二甲苯(或环保脱蜡剂)中10分钟；
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(或环保脱蜡剂)；
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；
6.取出玻片，再将其放入灭菌纯化水中室温洗涤3分钟，用无绒纸巾吸取多余水分；
7.取出玻片，再将玻片放入1×EDTA修复液中，高压锅高压修复2分钟(高压锅有气放出，开始发出响声时计时)；
8.冷却至室温后，取出玻片，自来水冲洗干净；
9.取出玻片，将其放入室温2×SSC中3分钟；
10.取出玻片，室温晾干；
11.将玻片正面朝上平放在架子上，在样本区域滴加适量的胃蛋白酶反应液， 室温消化5～15分钟；
12.将多余液体甩去，将其放入室温2×SSC中5分钟；
13.取出玻片，再将其放入另一缸室温2×SSC中5分钟；
14.取出玻片，再将其依次放入室温70％，90％，100％梯度乙醇脱水各2分钟；
15.取出玻片，室温晾干。
2、样品和探针同时变性(避光操作)
2.1从-20±5℃冰箱中取出杂交液，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22×22mm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。
4、结果分析
相关荧光和DAPI需用合适的滤块观察。
1.使用合适的滤镜，在10×物镜下寻找，在100×物镜下计数；
2.调整合适的焦距，对信号和背景有明确的概念；信号点应位于细胞内；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；
3.扫视几个细胞区域，要求细胞边界完整、无重叠，DAPI染色均匀，绿色和红色信号清晰；跳过信号弱及没有特定信号或高背景的细胞计数；需要主观辨别的细胞不计数；
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；
5.转到100×物镜，调整焦距，在核的不同层次找到所有信号点；
6.在每个细胞内计数信号点；调焦找到每个细胞内的所有信号点，计数一个区域内的两种或三种信号，只计数每种颜色有1个或更多FISH信号的，没有信号或只有单种颜色信号的细胞不计数；记录观察到的细胞总数(正常及异常信号数目)。
6.1正常信号：2F(ALK重排检测试剂盒)或2F2A(EML4/ALK重排检测试剂盒)；
6.2异常信号：1R1G1F(ALK重排检测试剂盒)
1F1A1G1RA(EML4/ALK重排检测试剂盒)。
7.设定阴性阈值
随机选取10例以上的样本当阴性质控片。每张阴性质控片随机计数信号完整的100个细胞，统计每例样本中出现异常信号细胞的数目及百分比，统计百分比的平均值及标准差，阴性阈值设定为平均值+3倍标准差。
实施例4：ALK基因和EML4基因检测试剂盒临床使用评价
使用实施例1所述ALK基因和EML4基因检测探针组合，实施例2所述2种检测试剂盒，一种为(ALK(组1)+ALK(组2)+EML4(组3)的组合(以下简称为EML4/ALK重排检测试剂盒)，以及另一种为ALK(组1)+ALK(组2)的组合的试剂盒(以下简称为ALK重排检测试剂盒)、对20份临床样本(其经过病理检测确诊，检测标本为福尔马林固定石蜡包埋组织样本，具体见下表)，分别进行检测。两种试剂盒的实验重复三次，检测结果 相同，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3A、图3B、图4A、图4B为两种基因三组探针组合试剂盒检测结果，其中，图3A为ALK重排检测试剂盒对肺癌组织样本FISH检测结果图，其中，检测信号类型为2F(RG相邻或重合)，ALK基因未重排。图3B为EML4/ALK重排检测试剂盒对肺癌组织样本FISH检测结果图，其中，检测信号类型为2F2A，ALK基因未重排，未发生EML4/ALK重排。图3B为实施例中肺癌组织样本FISH检测结果图，其中，检测信号类型为2F2A，ALK基因未重排，未发生EML4/ALK重排。图4A为ALK重排检测试剂盒对肺癌组织样本FISH检测结果图，其中，检测信号类型为1F1G1R，对比正常信号类型2F，发生了F信号断裂，检测结果为：ALK基因重排。图4B为EML4/ALK重排检测试剂盒对肺癌组织样本FISH检测结果图，其中，检测信号类型为1F1A1G1RA，对比正常信号类型2F2A，发生了F信号断裂，并同时发生RA信号融合，检测结果为：ALK基因断裂，重排类型为EML4/ALK。
本发明中，针对EML4基因和ALK基因的检测试剂盒，分别单独使用三组探针实现了基因重排、融合类型的检测。但相对探针组合使用而言，组合克隆探针的使用，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EML4基因的探针组3、ALK基因的探针组1和ALK基因的探针组2的检测探针的BAC克隆其长度分别为：261Kb、462Kb和513Kb。
ALK重排伙伴基因多，EML4/ALK重排是其中的一种类型。利用ALK基因重排检测探针(包含ALK两组探针)可以检测ALK重排，断裂即意味重排发生，但不能判断重排类型。利用EML4/ALK重排检测探针(包含ALK两组探针和EML4一组探针)可以检测ALK重排，并同时判断是否发生了EML4/ALK重排。
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。
Figure PCTCN2016105703-appb-000008
Figure PCTCN2016105703-appb-000009
从上述实验检测结果知，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。
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 (12)

  1. 一种ALK基因检测探针的制备方法，其特征在于，包括以下步骤：
    (1)选取针对ALK基因的BAC克隆为RP11-62B19、RP11-100C1和CTD-2245E6中至少一种作为一组探针，和选取BAC克隆为CTD-2544I11、RP11-684O3和RP11-134O13中的至少一种作为另一组探针；
    (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，同组探针的荧光素的颜色相同，不同组探针的荧光素的颜色不相同，即得。
  2. 根据权利要求1所述的ALK基因检测探针的制备方法，其特征在于，所述BAC克隆为RP11-62B19、RP11-100C1和CTD-2245E6，和所述BAC克隆为为CTD-2544I11、RP11-684O3和RP11-134O13。
  3. 根据权利要求1或2任一项所述的制备方法得到的ALK基因检测探针。
  4. 一种ALK基因检测试剂盒，其特征在于，包括有权利要求3所述的ALK基因检测探针。
  5. 一种ALK基因和EML4基因检测探针的制备方法，其特征在于，包括以下步骤：
    (1)选取针对ALK基因的BAC克隆为RP11-62B19、RP11-100C1和CTD-2245E6中至少一种，和选取BAC克隆为CTD-2544I11、RP11-684O3和RP11-134O13中的至少一种；以及选取针对EML4基因的BAC克隆为CTD-2358L8和CTD-2547J15中至少一种；
    (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，针对ALK基因和针对EML4基因的检测探针标记的荧光素的颜色不相同，且针对ALK基因中，同组检测探针的荧光素的颜色相同，不同组检测探针的荧光素的颜色不相同。
  6. 根据权利要求5所述的ALK基因和EML4基因检测探针的制备方法，其特征在于，所述ALK基因检测探针有两组，其中一组BAC克隆为RP11-62B19、RP11-100C1和CTD-2245E6；另一组BAC克隆为CTD-2544I11、 RP11-684O3和RP11-134O13。
  7. 根据权利要求5所述的ALK基因和EML4基因检测探针的制备方法，其特征在于，针对EML4基因检测探针的BAC克隆为CTD-2358L8和CTD-2547J15。
  8. 根据权利要求5-7任一项所述的ALK基因和EML4基因检测探针的制备方法，其特征在于，所述荧光素为Alexa
    Figure PCTCN2016105703-appb-100001
    FITC、Alexa
    Figure PCTCN2016105703-appb-100002
    Rhodamine、Texas Red、pacific
    Figure PCTCN2016105703-appb-100003
    DEAC。
  9. 根据权利要求5-7任一项所述ALK基因和EML4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，所述标记的温度为15℃-18℃，标记的时间为8-12小时。
  10. 根据权利要求5-9任一项所述的制备方法得到的ALK基因和EML4基因检测探针。
  11. 一种ALK基因和EML4基因检测试剂盒，其特征在于，包括有权利要求10所述的ALK基因和EML4基因检测探针。
  12. 根据权利要求11所述的ALK基因和EML4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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