WO2017114008A1 - Bcr基因和abl基因检测探针及其制备方法和试剂盒 - Google Patents

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

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WO2017114008A1
WO2017114008A1 PCT/CN2016/105715 CN2016105715W WO2017114008A1 WO 2017114008 A1 WO2017114008 A1 WO 2017114008A1 CN 2016105715 W CN2016105715 W CN 2016105715W WO 2017114008 A1 WO2017114008 A1 WO 2017114008A1
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ctd
bcr
abl
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何瑰
陈绍宇
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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  • the invention belongs to the biotechnology, in particular to a BCR gene and ABL gene detection probe, a preparation method thereof and a kit.
  • Chronic myelogenous leukemia is a clonal myeloproliferative neoplasm derived from hematopoietic stem cells, mainly involving the granulocyte cell line, which is characterized by an increase in the number of persistent, progressive peripheral blood leukocytes.
  • CML is slow onset and the initial symptoms are not obvious. The natural course of disease progresses from the chronic phase to the accelerated phase, and finally develops into a blast phase. After a sudden change, the patient often dies within 3 to 5 months.
  • Ph chromosome More than 90% of patients with leukemia cells have a constant, characteristic Ph chromosome and its molecular marker BCR/ABL fusion gene.
  • Complex translocation hidden translocation.
  • the Ph chromosome exists in the entire course of CML, and persists after treatment remission. Therefore, bone marrow transplantation and elimination of Ph-positive clones can achieve the final cure.
  • CML can be diagnosed and screened for tyrosine kinase inhibitor beneficiaries.
  • 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. , the hybrid signal is strong, Easy to detect; 2) Whole-chromosomal or chromosomal region-specific probes consisting of extremely different nucleotide segments on a chromosome or a segment of a chromosome, obtained from chromosome-specific large fragments cloned into phage and plasmid ; 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 a BCR gene and ABL gene detection probe and a preparation method thereof, and the prepared probe can be used for detecting the BCR gene and the ABL gene state, that is, detecting BCR gene and ABL gene detection, and detecting fusion.
  • Types include typical translocations, variant translocations, and concealed translocations with good specificity.
  • a method for preparing a BCR gene and an ABL gene detecting probe comprises the following steps:
  • BAC clone for the BCR gene as at least one of RP11-1026A5, RP11-165G5, CTD-2079I4, and selecting a BAC clone as at least one of CTD-2302P22, CTD-2037J11, CTD-2509L4;
  • the BAC clone targeting the ABL gene is at least one of CTD-2037L19, RP11-21G10, and CTD-2526G20;
  • the BAC clones against the ABL gene are CTD-2037L19, RP11-21G10, and CTD-2526G20.
  • the BAC clones for the BCR gene are RP11-1026A5, RP11-165G5, and CTD-2079I4, and the BAC clones are CTD-2302P22, CTD-2037J11, and CTD-2509L4.
  • 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 a BCR gene and ABL gene detecting kit.
  • a BCR gene and ABL gene detection kit includes the above BCR gene and ABL gene detection probe.
  • the present invention detects the BCR/ABL fusion gene of known or unknown type by using FISH (Fluorescence In-Situ Hybridization) method by screening the optimal BCR gene and ABL gene detection probes and their combinations; Identification of BCR fracture types.
  • FISH Fluorescence In-Situ Hybridization
  • BCR/ABL fusion detection by FISH method supplements the deficiency of BCR/ABL fusion detection in clinical, which is beneficial to accuracy Confirmed CML, improve patient survival and overall survival.
  • the preferred clone of the present invention has good detection specificity and high sensitivity. Through the visual detection of large segment rearrangement, it is not easy to miss the complex mutation type, and it also has a good discriminativeness for the unknown fusion type.
  • the BCR gene and the ABL kit according to the present invention can be applied in the fields of tumor biology and cytogenetics, and can comprehensively evaluate various molecular markers, and assist in the selection of clinical targeted therapeutic drugs and treatment options.
  • Figure 1A is a schematic illustration of the ABL gene detection probe sequence of Example 1.
  • Fig. 1B is a schematic diagram showing the BCR gene detecting probe sequence of Example 1.
  • Fig. 2 is a graph showing the results of FISH detection of the human peripheral blood culture cell sheet BCR gene and the ABL gene detection probe in Example 1.
  • Figure 3 is a graph showing the results of FISH detection of the clinical bone marrow samples in Example 4. The results show that 2R2GA has no BCR/ABL fusion.
  • Example 4 is a graph showing the results of FISH detection of clinical bone marrow samples in Example 4. The results show that 1R1GA/1RG1RA was found to be BCR/ABL fusion.
  • Figure 5 is a graph showing the results of FISH detection of the clinical bone marrow sample in Example 4, and the results show that 1R1GA/1RG1RGA, BCR/ABL Gene fusion, and is a secondary type of break.
  • the preparation method of the ABL detection probe comprises the following steps:
  • Cloning screening Clones containing the target gene ABL and the sequences at both ends were selected, as shown in Fig. 1A.
  • the GSP ABL1 includes a first probe, a second probe, a third probe, a fourth probe, and a fifth probe, which may be one, several, or a plurality of combinations.
  • ABL1 gene chr9 133, 589, 268-133, 763, 062, 173, 795 bp
  • Probe set 1 BAC Insert start and end position First probe CTD-2037L19 Chr9: 133263327...133469440 (206Kb) Second probe RP11-21G10 Chr9: 133463380...133642562 (179Kb) Third probe CTD-2526G20 Chr9: 133, 641, 402-133, 714, 138 (73Kb) Fourth probe RP11-818C20 Chr9: 133, 733, 184-133, 922, 526 (189Kb) Fifth probe RP11-728O24 Chr9: 133, 899, 288-134, 059, 021 (160Kb)
  • the preparation method of the BCR detection probe comprises the following steps:
  • Cloning Screening Clones containing the two-end sequences outside the BCR M-BCR site of the gene of interest were selected to prepare a GSP BCR probe, as shown in Figure 1B.
  • the GSP BCR includes two sets of probes located at the 3' and 5' ends of the BCR major breakpoint, respectively.
  • Probe Set 2 (Left: Green) BAC Insert start and end position
  • Probe set 3 (right: blue) BAC Insert start and end position Ninth probe CTD-2302P22 Chr22:23665325...23780116(115Kb) Tenth probe CTD-2037J11 Chr22:23780999...23921572(141Kb) Eleventh probe CTD-2509L4 Chr22:23938945...24160784(222Kb)
  • 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 cultured cells contain metaphase or interphase chromosomal DNA.
  • the chromosomal DNA appears as a morphologically recognizable chromosome or nucleus.
  • Figure 2 (experimental results applied to probe set 1 + probe set 2+ probe set 3) shows the results of FISH hybridization of metaphase chromosomes.
  • the BCR/ABL fusion gene detection probe signal is bright, and the sensitivity and specificity can be observed on the metaphase chromosome in the human peripheral blood culture cell sheet by 100%; the BCR and AB can be clearly recorded by using the bone marrow sample piece for hybridization detection.
  • the red signal indicates GSP ABL1
  • the green signal and cyan signal indicate the probes at both ends of the GSP BCR main break point, respectively.
  • the BCR gene and ABL gene detection kit includes two components of a BCR/ABL hybridization solution and a DAPI counterstain, wherein the BCR and ABL hybridization solution comprises the set of GSP ABL gene probes described in Example 1 and two sets of GSP BCRs. A combination of gene probes.
  • the BCR gene has two sets of detection probes, and the ABL gene has a set of detection probes.
  • the BCR gene and the ABL gene detection kit are: BCR (group 2+ group 3) + ABL (group 1) 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 cells are required to have no overlap, and the number of single-field cells is 100 ⁇ . 200 are appropriate.
  • the relevant fluorescence and DAPI need to be observed with a suitable filter block.
  • Cells with 50 to 200 cells counted, RG fusion, RA fusion, and RGA fusion signals were recorded as abnormal cells; cells with 2R2GA signal type were recorded as normal cells.
  • Example 3 Using the BCR gene and ABL gene detection probe combination described in Example 1, the detection kit described in Example 2 (ABL (Group 1) + BCR (Group 2) + BCR (Group 3) combination, 20 clinical samples (It was confirmed by pathological examination, see the table below), and the test was performed separately. The experiment of the two probe combinations was repeated three times, the test results were the same, and the test consistency was good. Compared with the commercially available reagents, the test results were completely consistent, the reagents were the same. The specificity and sensitivity are high. Figure 3 and Figure 4 show the results of the combination kit. As shown in Figure 3, the signal type is 1R2GA. Compared with the normal signal 2R2GA, the R signal is missing. Therefore, the result is judged as ABL gene deletion.
  • the signal type is 1R1GA/1RG1RA.
  • the GA signal is broken, the R signal is broken, and the GR and AR signals are merged at the same time.
  • the result was judged as BCR/ABL gene fusion and was the main type of fragmentation.
  • the signal type was 1R1GA/1RG1RGA.
  • the G signal was broken and the R signal was broken and simultaneously occurred.
  • GR, G AR signal fusion so the result is judged as BCR/ABL gene fusion, and Secondary break type.
  • the detection of the breakpoint and the fusion type is performed using three sets of probes, respectively.
  • the BAC clones of the probe set for the probe group 1 of the ABL1 gene, the probe set 2 of the BCR gene, and the probe set 3 of the BCR gene of the present invention have lengths of 796 Kb, 318 Kb, and 495 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

涉及BCR基因和ABL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BCR基因的BAC克隆为RP11-1026A5、RP11-165G5、CTD-2079I4中至少一种，和选取BAC克隆为CTD-2302P22、CTD-2037J11、CTD-2509L4中的至少一种；和选取针对ABL基因的BAC克隆为CTD-2037L19、RP11-21G10、CTD-2526G20中至少一种；得到质粒DNA后进行荧光标记。还公开了包含有BCR基因和ABL基因检测探针的试剂盒。

Description

BCR基因和ABL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BCR基因和ABL基因检测探针及其制备方法和试剂盒。
背景技术
慢性粒细胞白血病(chronic myelogenous leukemia,CML)是一种起源于造血干细胞的克隆性骨髓增殖性肿瘤，主要累及粒细胞系，表现为持续、进行性外周血白细胞数量增加。CML起病缓慢，初期症状不明显。其自然病程是由慢性期进展为加速期，最后发展为急变期，急变后患者常在3～5个月内死亡。
90％以上患者白血病细胞中有恒定的、特征性的Ph染色体及其分子标志BCR/ABL融合基因。Ph染色体绝大多数为t(9；22)(q34；q11)典型易位，少数患者有变异易位，包括22号染色体与非9号染色体间简单变异易位，3条或更多条染色体间的复杂易位(隐匿易位)。Ph染色体存在于CML的整个病程中，治疗缓解后，仍持续存在，因此采用骨髓移植，消除Ph阳性克隆，才能达到最终治愈。
因此，通过对初诊患者进行BCR/ABL检测，可以诊断CML，进行酪氨酸激酶抑制剂受益人群筛查。
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BCR/ABL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BCR基因和ABL基因检测探针及其制备方法，所制备的探针可用于检测BCR基因和ABL基因状态，即检测BCR基因和ABL基因检测，检测的融合类型包括典型易位、变异易位和隐匿易位，具有很好的特异性。
实现上述目的的技术方案如下。
一种BCR基因和ABL基因检测探针的制备方法，包括以下步骤：
(1)选取针对BCR基因的BAC克隆为RP11-1026A5、RP11-165G5、CTD-2079I4中至少一种，和选取BAC克隆为CTD-2302P22、CTD-2037J11、CTD-2509L4中的至少一种；和选取针对ABL基因的BAC克隆为CTD-2037L19、RP11-21G10、CTD-2526G20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BCR基因和针对ABL基因的检测探针标记的荧光素的颜色不相同，且针对BCR基因中，来源于BAC克隆RP11-1026A5、RP11-165G5、CTD-2079I4的探针与来源于BAC克隆CTD-2302P22、CTD-2037J11、CTD-2509L4的探针的荧光素的颜色不相同，即得。
在其中一个实施例中，针对ABL基因的所述BAC克隆为CTD-2037L19、RP11-21G10、和CTD-2526G20。
在其中一个实施例中，针对BCR基因的BAC克隆为RP11-1026A5、RP11-165G5、和CTD-2079I4，和BAC克隆为CTD-2302P22、CTD-2037J11、和CTD-2509L4。
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 PCTCN2016105715-appb-000001
FITC、Alexa
Figure PCTCN2016105715-appb-000002
Rhodamine、Texas Red、pacific
Figure PCTCN2016105715-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BCR基因和ABL基因检测试剂盒。
实现该目的技术方案如下。
一种BCR基因和ABL基因检测试剂盒，包括有上述BCR基因和ABL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BCR基因和ABL基因检测探针及其组合，采用FISH(Fluorescence In-Situ Hybridization)方法实现对已知或未知类型的BCR/ABL融合基因检测；可实现对BCR断裂类型的鉴定。
2.通过本发明所述的检测探针进行准确、快速的信号计数、且结果重复性好；通过FISH方法对BCR/ABL融合检测，补充了临床中BCR/ABL融合检测的不足，有利于准确确诊CML，提高患者生存率和总生存期。
3.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。
4.通过本发明所述的BCR基因和ABL试剂盒，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ABL基因检测探针序列的示意图。
图1B为是实施例1中BCR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BCR基因和ABL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，结果显示2R2GA，未见BCR/ABL融合。
图4为实施例4中临床骨髓样本FISH检测结果图，结果显示1R1GA/1RG1RA，发现BCR/ABL融合。
图5为实施例4中临床骨髓样本FISH检测结果图，结果显示1R1GA/1RG1RGA，BCR/ABL 基因融合，且为次要断裂类型。
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1BCR基因和ABL基因检测探针的制备
所述ABL检测探针(GSP ABL)的制备方法，包括以下步骤：
(1)克隆筛选：挑选包含目的基因ABL及两端序列的克隆，如图1A所示。
GSP ABL1包括第一探针、第二探针、第三探针、第四探针、第五探针，可以是其中的一个、几个，或者是多个组合。
本实施例选择5种探针的组合，具体如下表，其购买于Invitrogen RP11BAC及CTD BAC克隆库。
ABL1基因  chr9:133,589,268-133,763,062,173,795bp
探针组1 BAC 插入片段起止位置
第一探针 CTD-2037L19 chr9:133263327…133469440(206Kb)
第二探针 RP11-21G10 chr9:133463380…133642562(179Kb)
第三探针 CTD-2526G20 chr9:133,641,402-133,714,138(73Kb)
第四探针 RP11-818C20 chr9:133,733,184-133,922,526(189Kb)
第五探针 RP11-728O24 chr9:133,899,288-134,059,021(160Kb)
所述BCR检测探针(GSP BCR)的制备方法，包括以下步骤：
克隆筛选：挑选包含目的基因BCR M-BCR位点外的两端序列的克隆制备GSP BCR探针，如图1B所示。GSP BCR包括两组探针，这两组探针分别位于BCR主要断裂点的3`端和5`端。
BCR基因  chr22:23,522,552-23,660,224，137,673bp
探针组2(左：绿) BAC 插入片段起止位置
第六探针 RP11-1026A5 chr22：23384725…23567903(183Kb)
第七探针 RP11-165G5 chr22：23222620…23362028(139Kb)
第八探针 CTD-2079I4 chr22：23249861…23407965(158Kb)
探针组3(右：青) BAC 插入片段起止位置
第九探针 CTD-2302P22 chr22：23665325…23780116(115Kb)
第十探针 CTD-2037J11 chr22：23780999…23921572(141Kb)
第十一探针 CTD-2509L4 chr22：23938945…24160784(222Kb)
(2)GSP BCR和GSP ABL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BCR基因或ABL基因的3种或5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 BCR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 ABL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 PCTCN2016105715-appb-000004
Figure PCTCN2016105715-appb-000005
配完后震荡混匀，在16℃标记10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 BCR和GSP ABL基因探针，避光、-20℃储存。
(4)GSP BCR和GSP ABL基因探针验证：分别使用ABL探针组1+BCR探针组2+BCR探针组3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)。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2(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。图中可见BCR/ABL融合基因检测探针信号明亮，人外周血培养细胞片中在中期染色体上可观察到灵敏度、特异性100％；使用骨髓样本片进行杂交检测，可以清楚的记录BCR和AB合基因信号情况。红色信号表示GSP ABL1，绿色信号和青色信号分别表示GSP BCR主要断裂点两端探针。
实施例2：BCR基因和ABL基因检测试剂盒制备方法
BCR基因和ABL基因检测试剂盒包括有BCR/ABL杂交液和DAPI复染剂两个组分，其中BCR和ABL杂交液包含实施例1所述的一组GSP ABL基因探针和两组GSP BCR基因探针的组合。BCR基因有两组检测探针，ABL基因有一组检测探针，本实施例中，所述BCR基因和ABL基因检测试剂盒，为：BCR(组2+组3)+ABL(组1)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 PCTCN2016105715-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
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：BCR基因和ABL基因检测试剂盒的检测方法
1、样品处理
1.1取待检测外周血或骨髓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。
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；
4.2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；
4.3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。
5、杂交后洗涤及复染
5.1玻片放入72±1℃洗液I(1×SSC/0.3％NP-40)中2分钟；
5.2取出玻片，再将其放入室温洗液II(0.1％NP-40/2×SSC)中30秒；
5.3取出玻片，再将其放入室温70％，90％，100％乙醇中各2分钟；
5.4取出玻片，暗处自然干燥玻片；
5.5滴加10μl DAPI复染剂，待观察。
6、结果分析
相关荧光和DAPI需用合适的滤块观察。计数50～200个细胞，出现RG融合、RA融合、RGA融合信号的细胞记为异常细胞；出现2R2GA信号类型的细胞记为正常细胞。
实施例4：BCR基因和ABL基因检测试剂盒临床使用评价
使用实施例1所述BCR基因和ABL基因检测探针组合，实施例2所述检测试剂盒(ABL(组1)+BCR(组2)+BCR(组3)的组合、对20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。两种探针组合的实验重复三次，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3和图4为组合试剂盒检测结果，图3中所示，信号类型表现为1R2GA，与正常信号2R2GA比较，出现R信号缺失，所以，结果判断为ABL基因缺失，未发生BCR/ABL基因融合；图4中所示，信号类型表现为1R1GA/1RG1RA，与正常信号2R2GA比较，出现了GA信号断裂，R信号断裂，并同时发生了GR,AR信号融合，所以，结果判断为BCR/ABL基因融合，且为主要断裂类型。图5中所示，信号类型表现为1R1GA/1RG1RGA，与正常信号2R2GA比较，出现了G信号断裂，R信号断裂，并同时发生了GR,GAR信号融合，所以，结果判断为BCR/ABL基因融合，且为 次要断裂类型。
本发明中，针对ABL基因和BCR基因，分别使用三组探针实现了断裂点、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ABL1基因的探针组1、BCR基因的探针组2和BCR基因的探针组3的检测探针的BAC克隆其长度分别为：796Kb、318Kb和495Kb。
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 PCTCN2016105715-appb-000007
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BCR基因和ABL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BCR基因的BAC克隆为RP11-1026A5、RP11-165G5、CTD-2079I4中至少一种，和选取BAC克隆为CTD-2302P22、CTD-2037J11、CTD-2509L4中的至少一种；和选取针对ABL基因的BAC克隆为CTD-2037L19、RP11-21G10、CTD-2526G20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BCR基因和针对ABL基因的质粒DNA标记的荧光素的颜色不相同，且针对BCR基因中，来源于BAC克隆RP11-1026A5、RP11-165G5、CTD-2079I4的质粒DNA与来源于BAC克隆CTD-2302P22、CTD-2037J11、CTD-2509L4的质粒DNA的荧光素的颜色不相同，即得。
  2. 根据权利要求1所述的BCR基因和ABL基因检测探针的制备方法，其特征在于，针对ABL基因的所述BAC克隆为CTD-2037L19、RP11-21G10、和CTD-2526G20。
  3. 根据权利要求1所述的BCR基因和ABL基因检测探针的制备方法，其特征在于，针对BCR基因的BAC克隆为RP11-1026A5、RP11-165G5、和CTD-2079I4，和为BAC克隆为CTD-2302P22、CTD-2037J11、和CTD-2509L4。
  4. 根据权利要求1所述的BCR基因和ABL基因检测探针的制备方法，其特征在于，所述荧光素为Alexa
    Figure PCTCN2016105715-appb-100001
    FITC、Alexa
    Figure PCTCN2016105715-appb-100002
    Rhodamine、Texas Red、pacific
    Figure PCTCN2016105715-appb-100003
    或DEAC。
  5. 根据权利要求1-4任一项所述BCR基因和ABL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BCR基因和ABL基因检测探针。
  7. 一种BCR基因和ABL基因检测试剂盒，其特征在于，包括有权利要求6所述的BCR基因和ABL基因检测探针。
  8. 根据权利要求7所述的BCR基因和ABL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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