WO2017114006A1 - Aml1基因和eto基因检测探针及其制备方法和试剂盒 - Google Patents

Aml1基因和eto基因检测探针及其制备方法和试剂盒 Download PDF

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WO2017114006A1
WO2017114006A1 PCT/CN2016/105713 CN2016105713W WO2017114006A1 WO 2017114006 A1 WO2017114006 A1 WO 2017114006A1 CN 2016105713 W CN2016105713 W CN 2016105713W WO 2017114006 A1 WO2017114006 A1 WO 2017114006A1
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aml1
eto
ctd
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陈绍宇
何瑰
张会清
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广州安必平医药科技股份有限公司
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Definitions

  • Cytogenetic analysis is the most commonly used method for detecting t(8;21). However, for hidden translocation or complex translocation forms, it is easy to cause missed detection.
  • the PCR method can only detect gene sequences in a small range, so when the gene sequence is mutated, it is prone to false negative results.
  • the FISH assay can be used for detection of AML1/ETO fusion genes in peripheral blood or intermedullary cells. It is sensitive, specific, efficient and reproducible. It can reveal traditional cytogenetic analysis or by blood or bone marrow morphology. Submicroscopic anomalies were not detected in the examination.
  • interphase FISH can be used for the diagnosis of acute myeloid leukemia t(8;21)(q22;q22) (AML1/ETO).
  • AML1/ETO acute myeloid leukemia t(8;21)(q22;q22)
  • foreign countries have made great achievements in the detection of AML1/ETO fusion genes, while domestically limited by various conditions, detection methods are lagging, lack of corresponding molecular diagnostic reagents, diagnostic reagents rely on imports, high reagent prices, high detection costs, and difficult to promote .
  • 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.
  • One of the objects of the present invention is to provide an AML1 gene and an ETO gene detecting probe and a preparation method thereof, which can be used for detecting the AML1 gene and the ETO gene state, that is, detecting the AML1 gene and the ETO gene detection, and realizing the cell and Direct observation of signals in chromosomes has good specificity.
  • the BAC clone targeting the AML1 gene is at least one of CTD-3245J1, RP11-77G18, CTD-2349F18, CTD-3171K21 and RP11-384N13, and the BAC clones for the ETO gene are selected as RP11-1107H4, RP11- At least one of 302P1, CTD-2547C5, RP11-55J5, and CTD-2079N17;
  • the BAC clones of the AML1 probe are CTD-3245J1, RP11-77G18, CTD-2349F18, CTD-3171K21, and RP11-384N13.
  • the BAC clones of the ETO probe are RP11-1107H4, RP11-302P1, CTD-2547C5, RP11-55J5, and CTD-2079N17.
  • the labeled fluorescein selects a fluorescent dye known in the art, preferably fluorescein is Alexa FITC, Alexa Rhodamine, Texas Red, pacific DEAC.
  • the temperature of the label is between 15 ° C and 18 ° C and the time of labeling is between 8 and 12 hours.
  • An AML1 gene and ETO gene detection kit comprising the above AML1 gene and ETO gene detection probe.
  • the present invention detects the AML1/ETO fusion gene by FISH (Fluorescence In-Situ Hybridization) by screening the optimal AML1/ETO fusion gene detection probe and its combination, and the signal counting is accurate and rapid, and the result is obtained.
  • FISH Fluorescence In-Situ Hybridization
  • the reproducibility is good; supplementing the lack of import-dependent AML1/ETO fusion detection in clinical, it is beneficial to screen more patients who benefit from targeted drugs, and improve the survival rate and overall survival of patients with acute non-lymphocytic leukemia.
  • Figure 1B is a schematic illustration of the ETO gene detection probe sequence of Example 1.
  • Fig. 2 is a graph showing the results of FISH detection of the human peripheral blood culture cell sheet AML1 gene and ETO gene detection probe in Example 1.
  • Example 3 is a diagram showing the results of FISH detection of a clinical bone marrow sample in Example 4, wherein the detection signal type is 2R2G, and the AML1/ETO fusion gene detection is negative.
  • Example 4 is a diagram showing the results of FISH detection of a clinical bone marrow sample in Example 4, wherein the detection signal type is 1R1G2F, and the AML1/ETO fusion gene detection is positive.
  • the preparation method of the AML1/ETO detection probe of the present embodiment comprises the following steps:
  • GSP AML1 includes a first probe, a second probe, a third probe, and a fourth probe and a fifth probe, as shown in the following table, which were purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • the GSP ETO includes a first probe, a second probe, a third probe, a fourth probe, and a fifth probe, as shown in the following table, which was purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • the plasmid DNA mixture was fluorescently labeled by a nick translation method, and the fluorescein labeled for each probe of the ETO gene was Spectrum-Orange, and the fluorescein labeled for each probe of the AML1 gene was Spectrum-Green dUTP.
  • 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 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:
  • the AML1 and ETO gene detection kits include two components of AML1 and ETO hybridization solution and DAPI counterstaining agent, wherein the AML1 and ETO hybridization solution comprises the GSP AML1 and GSP ETO gene probes described in Example 1 for use in a hybrid environment. Buffer components (promoting hybridization), COT Human DNA blocking the repeat, 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 cells have no overlap, and the number of single-field cells is preferably from 100 to 200.
  • step 3 If the cell density and number are appropriate, proceed to step 3;
  • the cell drops can be placed in a closed container filled with absolute ethanol and stored at -20 ⁇ 5 ° C for one year.
  • the remaining cell suspension can be stored at 2-8 ° C for one month to allow for re-production if necessary.
  • the prepared lotion I was placed in a 72 ⁇ 1 ° C water bath, and the lotion II was placed at room temperature;
  • GSP AML1 probe shows green signal
  • GSP ETO probe is red signal
  • More than 5 human peripheral blood cells or bone marrow cells were randomly selected and processed according to the sample processing requirements to prepare a negative threshold reference sheet. Each reference piece was randomly counted for 200 cells. Observe the green (AML1) and red (ETO) signal points in each core. When the red and green signal points overlap or the proximity distance is less than one signal diameter, it is recorded as 1 fused signal (yellow, F); otherwise, it is recorded as 1 red (R) and 1 green (G) signal.
  • Fig. 3 and Fig. 4 show the detection results of the kits of the two sets of probes.
  • the type of the detection signal is 2R2G, which is expressed as a normal signal type. Therefore, the result is judged to be negative for the AML1/ETO fusion gene detection.
  • the detection signal type is 1R1G2F.
  • the R and G color signals are normal (normal chromosomes), and the other group of red R signals are broken.
  • the green G signal also ruptured, and the R and G signal fusions occurred at the same time. Therefore, the result was judged to be positive for the AML1/ETO fusion gene.
  • gene state and fusion detection are performed using the two sets of probes for the AML1 gene and the ETO gene, respectively, but the detection signal is better for the use of the combined clone probe in combination with the probe.
  • 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 length of the BAC clone of the probe set for the AML1 gene and the probe set of the ETO gene of the present invention are 878 Kb and 791 Kb, respectively.
  • 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

一种AML1基因和ETO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AML1基因的BAC克隆为CTD-3245J1、RP11-77G18、CTD-2349F18、CTD-3171K21和RP11-384N13中的至少一种，和选取针对ETO基因的BAC克隆为RP11-1107H4、RP11-302P1、CTD-2547C5、RP11-55J5和CTD-2079N17中的至少一种；对克隆分别提取质粒，得到质粒DNA；用荧光素标记质粒DNA。本发明还提供了包含AML1基因和ETO基因检测探针的试剂盒。

Description

AML1基因和ETO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AML1基因和ETO基因检测探针及其制备方法和试剂盒。
背景技术
急性髓系白血病t(8；21)(q22；q22)(AML1/ETO)是一种通常表现为中性粒细胞伴有成熟分化的AML，是AML最常见的染色体结构异常之一，占AML的5％～12％；占核型异常伴成熟之AML的1/3。约有8％成人和12％儿童急性髓系白血病患者可见该融合基因，其中以AML M2型(50％)和AML M2b(90％)型多见。t(8；21)(q22；q22)易位影响AML1基因(又称RUNX1)，此基因编码CBFa及ETO基因。基因转录物在AML伴t(8；21)患者中可检测到。AML1基因的断裂发生于单个内含子内部。
AML1/ETO融合被认为是一种预后良好的细胞和分子遗传学异常，伴有t(8；21)的AML通常化疗反应好，缓解率高，巩固期使用大剂量阿糖胞苷(HD Ara-C)无病生存期长。但若伴有其他分子学异常，如一条性染色体缺失及del(9)(q22)、KIT、FLT3、AML1-ETO9a剪接变体等更易致白血病生成，且影响预后。AML1-ETO能抑制GATA-1乙酰化，从而抑制红系分化，AML1/ETO对其靶基因的影响以及冬凌草、格列卫靶向治疗已成为目前研究的热点。
细胞遗传学分析(核型分析)是最常使用的检测t(8；21)的方法。但对于隐匿易位或复杂易位形式，易造成漏检。PCR方法只能检测很小范围内的基因序列，所以当基因序列发生变异时，易出现假阴性结果。FISH检测方法可用于外周血或骨髓间期细胞中AML1/ETO融合基因检测，具有敏感，特异，高效和重复性好的特点，它能揭示了传统的细胞遗传学分析或由血液或骨髓形态学检查均未检出亚微观异常。因此，间期FISH可用于急性髓系白血病t(8；21)(q22；q22)(AML1/ETO)诊断。目前，国外在AML1/ETO融合基因检测方面取得很大成就，而国内受各方面条件限制，检测方法滞后，缺少相应的分子诊断试剂，诊断试剂依赖进口，试剂价格高，检测成本高，不易推广。
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AML1/ETO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AML1基因和ETO基因检测探针及其制备方法，所制备的探针可用于检测AML1基因和ETO基因状态，即检测AML1基因和ETO基因检测，实现细胞和染色体中直接观察信号，具有很好的特异性。
实现上述目的的技术方案如下。
一种AML1基因和ETO基因检测探针的制备方法，包括以下步骤：
(1)选取针对AML1基因的BAC克隆为CTD-3245J1、RP11-77G18、CTD-2349F18、CTD-3171K21和RP11-384N13中至少一种，和选取针对ETO基因的BAC克隆为RP11-1107H4、RP11-302P1、CTD-2547C5、RP11-55J5和CTD-2079N17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，针对同一种基因的质粒DNA所标记的荧光素相同，针对 AML1基因和针对ETO基因的检测探针标记的荧光素的颜色不相同，即得。
在其中一个实施例中，所述AML1探针的BAC克隆为CTD-3245J1、RP11-77G18、CTD-2349F18、CTD-3171K21和RP11-384N13。
在其中一个实施例中，所述ETO探针的BAC克隆为RP11-1107H4、RP11-302P1、CTD-2547C5、RP11-55J5和CTD-2079N17。
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 PCTCN2016105713-appb-000001
FITC、Alexa
Figure PCTCN2016105713-appb-000002
Rhodamine、Texas Red、pacific
Figure PCTCN2016105713-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AML1基因和ETO基因检测试剂盒。
实现该目的技术方案如下。
一种AML1基因和ETO基因检测试剂盒，包括有上述AML1基因和ETO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AML1/ETO融合基因检测探针及其组合，采用FISH(Fluorescence In-Situ Hybridization)方法对AML1/ETO融合基因检测，信号计数行准确、快速，且结果的重复性好；补充了临床中AML1/ETO融合检测依赖进口的不足，有利于筛选更多受益于靶向药物的患者，提高急性非淋巴细胞白血病患者生存率和总生存期。
(2)本发明优选克隆检测特异性好，灵敏度高。通过对大片段重排的直观检测，不易遗漏复杂变异类型，对未知融合类型也有很好的鉴别性。
(3)通过本发明所述的急性非淋巴细胞白血病AML1/ETO融合试剂盒，从基因水平了解AML1/ETO融合状态改变，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 临床靶向治疗用药及治疗方案选择。
附图说明
图1A为是实施例1中AML1基因检测探针序列的示意图。
图1B为是实施例1中ETO基因检测探针序列的示意图。
图2为实施例1中人外周血培养细胞片AML1基因和ETO基因检测探针FISH检测结果图。
图3为实施例4中临床骨髓样本FISH检测结果图，其中，检测信号类型为2R2G，AML1/ETO融合基因检测阴性。
图4为实施例4中临床骨髓样本FISH检测结果图，其中，检测信号类型为1R1G2F，AML1/ETO融合基因检测阳性。
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1AML1基因和ETO基因检测探针的制备
本实施所述AML1/ETO检测探针的制备方法，包括以下步骤：
(1)挑选包含目的基因AML1和ETO及两端序列的克隆，如图1和2所示。
GSP AML1包括第一探针、第二探针、第三探针和第四探针和第五探针，具体如下表，其购买于Invitrogen RP11BAC及CTD BAC克隆库。
GSP ETO包括第一探针、第二探针、第三探针、第四探针和第五探针，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。
AML1
  BAC 插入片段起止位置
第一探针 CTD-3245J1 (chr21:35875546..36108830)(233Kb)
第二探针 RP11-77G18 (chr21:36106471..36280467)(174Kb)
第三探针 CTD-2349F18 (chr21:36258305..36361730)(103Kb)
第四探针 CTD-3171K21 (chr21:36354553..36566542)(211Kb)
第五探针 RP11-384N13 (chr21:36592109..36753937)(162Kb)
ETO
  BAC 插入片段起止位置
第一探针 RP11-1107H4 (chr8:92632470…92842840)(210Kb)
第二探针 RP11-302P1 (chr8:92824111…93023704)(200Kb)
第三探针 CTD-2547C5 (chr8:93004274…93198844)(195Kb)
第四探针 RP11-55J5 (chr8:93158871…93335264)(176Kb)
第五探针 CTD-2079N17 (chr8:93331140…93423658)(93Kb)
(2)基因探针的制备：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，最后分别将得到针对AML1基因和ETO基因的5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，针对ETO基因的每种探针标记的荧光素为Spectrum-Orange，针对AML1基因的每种探针标记的荧光素为Spectrum-Green 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 PCTCN2016105713-appb-000004
Figure PCTCN2016105713-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300bp左右存在弥散的带。
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 AML1和GSP ETO基因探针，避光、-20℃储存。
(4)GSP AML1和GSP ETO基因探针验证：使用AML1探针+ETO探针制备的杂交液，使用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)。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3所示：中期染色体的FISH杂交结果图。图中可以看见染色体相应位置显示红色荧光和绿色荧光信号。红色信号表示GSP ETO，绿色信号表示GSP AML1。
实施例2：AML1和ETO基因检测试剂盒制备方法
AML1和ETO基因检测试剂盒包括有AML1和ETO杂交液和DAPI复染剂两个组分，其中AML1和ETO杂交液包含实施例1所述的GSP AML1和GSP ETO基因探针、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：
杂交液配制
Figure PCTCN2016105713-appb-000006
Figure PCTCN2016105713-appb-000007
a.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。
b.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：AML1/ETO基因检测试剂盒的检测方法
1、样品处理
1.1取外周血或骨髓2～3ml(肝素钠抗凝)2000rpm离心5min，小心去上清。
1.2加入10ml的低渗液(0.075mol/L KCl),吹打混匀，静置3min。
1.3 37±1℃水浴箱低渗30min。
1.4加新鲜固定液1ml，吹打混匀，室温预固定10min。
1.5吹打混匀，2000rpm离心5min。
1.6去上清，沉淀加新鲜固定液5～10ml，吹打混匀，室温静置10min。
1.7 2000rpm离心5min，去上清。
1.8可重复以上洗涤步骤，直至细胞沉淀洗白洗干净(此步骤不需室温静置10min)。
2、制片
2.1取一张干净的载玻片；
2.2重悬细胞后取3μl悬液滴加到载玻片上；
2.3室温下晾干；
2.4用10×物镜在相差显微镜下观察细胞密度，要求细胞无重叠，且单视野细胞数量在100～200个为宜。
a)如果细胞密度及数目合适，继续步骤3；
b)如果细胞有重叠，则加入适量新鲜固定液稀释细胞悬液，混匀后另取3μl悬液制片，
c)如果细胞密度低，则2000rpm离心5min，小心吸去适量上清液，混匀后另取3μl悬液制片，晾干，观察；
2.5在相差显微镜下观察，如果细胞碎片太多，则需要做预处理并且选择合适的杂交区域；
注意:每个病例至少需要多制一张片，细胞滴片可置于放有无水乙醇的密闭容器中，在-20±5℃可以保存一年。剩余的细胞悬液可以在2～8℃保存一个月，以便必要时重新制片。
3、玻片预处理
3.1将滴好的玻片置于室温2×SSC(PH7.0)溶液中浸泡2min；
3.2依次在室温70％，90％，100％的乙醇中浸泡2min脱水；然后取出玻片，室温晾干。
4、样品和探针同时变性
4.1从-20℃冰箱中取出杂交液，震荡混匀，瞬时离心；
4.2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；
4.3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；
4.4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78℃2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)。
5、杂交后洗涤及复染
5.1洗涤前30分钟，将配制好的洗液I放入72±1℃水浴中,洗液II室温放置；
5.2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)；
5.3玻片放入72±1℃洗液I(1×SSC/0.3％NP-40)中2分钟；
5.4取出玻片，再将其放入室温洗液II(0.1％NP-40/2×SSC)中30秒；
5.5取出玻片，再将其放入室温70％，90％，100％乙醇中各2分钟；
5.6取出玻片，暗处自然干燥玻片；
5.7滴加10μl DAPI复染剂至干燥的22x22mm盖玻片上，反转样本片，使盖玻片与载玻片的目标区域接触，反转后轻压，避免产生气泡，在暗处存放，待观察。
6、结果分析
相关荧光和DAPI需用合适的滤块观察。其中，GSP AML1探针显示绿色信号；GSP ETO探针为红色信号。
6.1使用合适的滤镜，在10×物镜下寻找，在100×物镜下计数；
6.2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；
6.3扫视几个细胞区域，要求细胞核边界完整，DAPI染色均匀、核无重叠，绿色和红色信号点清晰；跳过信号弱及没有特定信号或高背景的核计数；需要主观辨别的核不计数；
6.4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；
6.5转到100×物镜，调整焦距，在核的不同层次找到所有信号点；
6.6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(信号数目正常及异常)；
6.7设定阴性阈值。
随机选取5例以上的人外周血细胞或骨髓细胞，按照样本处理要求进行处理，制备阴性阈值参考片。每张参考片随机计数200个细胞。观察每个核内的绿色(AML1)和红色(ETO)信号点。当红色与绿色信号点重叠在一起或两者临近距离小于一个信号直径时记为1个融合信号(黄色，F)；否则记为1个红色(R)和1个绿色(G)信号。
实施例4：AML1基因和ETO基因检测试剂盒临床使用评价
使用实施例1所述AML1基因和ETO基因检测探针组合，实施例2所述2组检测探针试剂盒，对18份临床样本(其经过病理检测确诊，具体见下表)，分别进行检测。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为两组探针的试剂盒的检测结果，图3中所示，检测信号类型为2R2G，表现为正常信号类型，因此，结果判断为AML1/ETO融合基因检测阴性。图4中，检测信号类型为1R1G2F，相对于正常信号类型2R2G，除了一组染色体R和G色信号表现正常(正常染色体)，另一组红色R信号出现断裂， 绿色G信号也出现断裂，并同时发生R和G信号融合，因此，结果判断为AML1/ETO融合基因检测阳性。
本发明中，针对AML1基因和ETO基因，分别使用两组探针实现了基因状态和融合检测，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AML1基因的探针组、ETO基因的探针组检测探针的BAC克隆其长度分别为：878Kb和791Kb。
Figure PCTCN2016105713-appb-000008
Figure PCTCN2016105713-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 (8)

  1. 一种AML1基因和ETO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AML1基因的BAC克隆为CTD-3245J1、RP11-77G18、CTD-2349F18、CTD-3171K21和RP11-384N13中至少一种，和选取针对ETO基因的BAC克隆为RP11-1107H4、RP11-302P1、CTD-2547C5、RP11-55J5和CTD-2079N17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，针对同一种基因的质粒DNA所标记的荧光素相同，针对AML1基因和针对ETO基因的检测探针标记的荧光素的颜色不相同，即得。
  2. 根据权利要求1所述的AML1基因和ETO基因检测探针的制备方法，其特征在于，针对ETO基因的所述BAC克隆为RP11-1107H4、RP11-302P1、CTD-2547C5、RP11-55J5和CTD-2079N17。
  3. 根据权利要求1所述的AML1基因和ETO基因检测探针的制备方法，其特征在于，针对AML1基因的BAC克隆为CTD-3245J1、RP11-77G18、CTD-2349F18、CTD-3171K21和RP11-384N13。
  4. 根据权利要求1所述的AML1基因和ETO基因检测探针的制备方法，其特征在于，所述荧光素为Alexa
    Figure PCTCN2016105713-appb-100001
    FITC、Alexa
    Figure PCTCN2016105713-appb-100002
    Rhodamine、Texas Red、pacific
    Figure PCTCN2016105713-appb-100003
    或DEAC。
  5. 根据权利要求1-4任一项所述AML1基因和ETO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。
  6. 根据权利要求1-5任一项所述的制备方法得到的AML1基因和ETO基因检测探针。
  7. 一种检测急性非淋巴细胞白血病AML1和ETO融合基因的试剂盒，其特征在于，包括有权利要求6所述的AML1基因和ETO基因检测探针。
  8. 根据权利要求7所述的急性非淋巴细胞白血病AML1和ETO融合基因的试剂盒，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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