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WO2017114011A1 - Her-2基因和/或top2a基因检测探针及其制备方法和试剂盒 - Google Patents

Her-2基因和/或top2a基因检测探针及其制备方法和试剂盒 Download PDF

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WO2017114011A1
WO2017114011A1 PCT/CN2016/105718 CN2016105718W WO2017114011A1 WO 2017114011 A1 WO2017114011 A1 WO 2017114011A1 CN 2016105718 W CN2016105718 W CN 2016105718W WO 2017114011 A1 WO2017114011 A1 WO 2017114011A1
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top2a
ctd
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bac
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陈绍宇
何瑰
席影
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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 a HER-2 (ERBB2) gene and/or TOP2A gene detecting probe, a preparation method thereof and a kit.
  • HER-2 ERBB2
  • TOP2A TOP2A gene detecting probe
  • HER-2 The HER-2 (ERBB2) gene has gene amplification and protein overexpression in 20%-30% of breast cancer patients.
  • HER-2 is currently recognized as an important prognostic/predictor for breast cancer.
  • HER-2 is an independent prognostic factor for tumor recurrence and overall survival.
  • HER-2 positive breast cancer is invasive, with short disease-free survival and poor prognosis.
  • Trastuzumab (Herceptin) is indicated for breast cancer with HER-2 overexpression and gene amplification.
  • TOP2A gene regulates the supercoiled state of DNA, directly participates in cellular processes, and is a target enzyme of anthracycline antibiotics.
  • the HER-2 gene is located in the 17q12 region of chromosome 17, the abnormality of HER-2 is amplified; the TOP2A gene is located in the 17q21 region of chromosome 17, and the abnormality of TOP2A is divided into amplification and deletion, and FISH detection can be used for this. A clear judgment is made in both cases.
  • 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. , hybrid signal is strong, easy to detect; 2) whole chromosome or chromosomal region-specific probe, which is on a chromosome or a segment on a chromosome Consisting of extremely different nucleotide fragments, which can be obtained from chromosome-specific large fragments cloned into phage and plasmids; 3) specific position probes 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 HER-2 gene and/or TOP2A gene detecting probe and a preparation method thereof, which can be used for detecting the HER-2 gene and the TOP2A gene state, that is, detecting the HER-2 gene. And the change in the copy number of the TOP2A gene has good specificity.
  • a method for preparing a HER-2 gene and/or a TOP2A gene detection probe comprising the steps of:
  • the BAC clone targeting the HER-2 gene is at least one of RP11-1021P11, RP11-62N23, and RP11-1044P23, or the BAC clone is at least one of RP11-98J2, RP11-1065L22, and CTD-2327I15. And/or selecting a BAC clone for the TOP2A gene as at least one of RP11-1152A10, CTD-3217C5, or selecting a BAC clone as at least one of RP11-737D6, CTD-3087O22, RP11-48O10, CTD-2134K5;
  • the plasmid DNA is labeled with fluorescein, and the fluorescein labeled with the plasmid DNA of the same gene is the same, and the color of the fluorescein labeled with the HER-2 gene and the detection probe for the TOP2A gene is different.
  • the BAC clones for the TOP2A gene are RP11-1152A10 and CTD-3217C5.
  • the BAC clones for the TOP2A gene are RP11-737D6, CTD-3087O22, RP11-48O10, and CTD-2134K5.
  • the BAC clones directed against the HER-2 gene are RP11-1021P11, RP11-62N23, and RP11-1044P23.
  • the BAC clones for the TOP2A gene are RP11-98J2, RP11-1065L22, and CTD-2327I15.
  • 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.
  • the label has a temperature of from 24 ° C to 26 ° C and a marked time of from 4 to 6 hours.
  • Another object of the present invention is to provide a HER-2 gene and TOP2A gene detecting kit.
  • a HER-2 gene and/or TOP2A gene detection kit comprising the above HER-2 gene and/or TOP2A gene detection probe.
  • a chromosome 17 discrimination probe (CSP17) probe for internal control is included, the identification probe being different in color from the fluorescein labeled by the HER-2 gene and the TOP2A gene detection probe.
  • CSP17 chromosome 17 discrimination probe
  • the present invention uses the FISH (Fluorescence In-Situ Hybridization) method to detect the copy number of the HER-2 gene and the TOP2A gene by screening the optimal HER-2 gene and/or TOP2A gene detection probes and their combinations.
  • FISH Fluorescence In-Situ Hybridization
  • the preferred clone of the present invention has good detection specificity and high sensitivity.
  • the length probe is limited to about 500 bp, which improves the hybridization efficiency and reduces the hybridization background.
  • the signal counting line is accurate and fast, and the result is reproducible; through the simultaneous detection and evaluation of HER-2, TOP2A copy number and chromosome 17, it is beneficial to determine the treatment plan and screen more breasts that benefit from targeted drugs. Cancer patients improve patient survival and overall survival.
  • the HER-2 gene and TOP2A kit of the present invention the HER-2 gene and TOP2A state changes are known from the gene level, and various signal types exhibit tumor cell genetic diversity of solid tissues, which can be realized in tumor organisms.
  • the application in the fields of learning and cytogenetics can help comprehensively evaluate various molecular markers, assist breast cancer prognosis and clinical targeted therapy drugs and treatment options.
  • Figure 1A is a schematic illustration of the HER-2 gene detection probe sequence of Example 1.
  • Fig. 1B is a schematic diagram showing the TOP2A gene detecting probe sequence of Example 1.
  • Fig. 2 is a graph showing the results of FISH detection of the HER-2 gene and the TOP2A gene detecting probe of human peripheral blood culture cell sheets in Example 1.
  • Example 3 is a diagram showing the results of FISH detection of breast cancer tissue samples in Example 4, wherein the detection signal type is 2R2G2A, the HER-2 gene is not amplified, and the TOP2A gene is not amplified.
  • Example 4 is a diagram showing the results of FISH detection of a breast cancer tissue sample in Example 4, wherein the detection signal type is clustered R cluster G2A, and the HER-2 gene and the TOP2A gene are co-amplified.
  • a method for preparing a HER-2 detection probe comprising the following steps:
  • Cloning screening Clones containing the target gene HER-2 and the sequences at both ends were selected, as shown in Fig. 1A.
  • GSP HER-2 consists of two groups, and the following two sets of detection probes are prepared separately.
  • HER-2 chr17 37, 856, 254-37, 884, 915, 28, 662 bp
  • HER-2 probe set 1 BAC Insert start and end position First probe RP11-1021P11 Chr17:37497503...37725641(228Kb) Second probe RP11-62N23 Chr17:37725641...37882864(157Kb) Third probe RP11-1044P23 Chr17:37871783...38066611(195Kb) HER-2 probe set 2 BAC Insert start and end position Fourth probe RP11-98J2 Chr17:37572511...37753435 (180Kb) Fifth probe RP11-1065L22 Chr17:37758567...37990799(232Kb) Sixth probe CTD-2327I15 Chr17:37963139...38082538(119Kb)
  • the preparation method of the TOP2A detection probe comprises the following steps:
  • Cloning Screening Clones containing the TOP2A gene of interest and the sequences at both ends were selected, as shown in Figure 1B.
  • GSP TOP2A consists of two groups, as shown in the following table, which were purchased from the Invitrogen RP11BAC and CTD BAC clone libraries. The following two sets of detection probes were separately prepared.
  • TOP2A chr17 38, 544, 773-38, 574, 202, 29, 430 bp
  • the plasmid DNA mixture was fluorescently labeled by the nick translation method.
  • the fluorescein labeled for each probe of the GSP HER-2 gene was Spectrum-Orange, and the fluorescein labeled for each probe of the GSP TOP2A gene was Spectrum- Green.
  • 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:
  • GSP HER-2 and GSP TOP2A gene probe validation hybridization solution prepared using HER-2 probe set 1 + TOP2A probe set 1 and HER-2 probe set 2+ TOP2A probe set 2, respectively
  • the hybridization solution is a reagent to be detected, and probe verification is performed using 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.
  • Figure 2 (experimental results applied to the HER-2 probe set 1 + TOP2A probe set 1) shows the results of FISH hybridization of metaphase chromosomes.
  • the red signal shows GSP HER-2
  • the green signal shows GSP TOP2A
  • the cyan signal shows CSP17 (for positioning the chromosome 17 centromere probe, purchased from D17Z1SpectrumAqua Probe, article number: 06J38-017).
  • the HER-2/TOP2A/17 chromosome probe signal is bright, and sensitivity and specificity can be observed on the metaphase chromosome in human peripheral blood cultured cells. 100% can be clearly recorded by using paraffin sample tablets for hybridization detection.
  • Three target copy numbers The results of other corresponding probe verification experiments are the same as those of the HER-2 probe set 1 + TOP2A probe set 2, and the figures are omitted.
  • the HER-2 gene and TOP2A gene detection kit includes two components of HER-2/TOP2A hybridization solution and DAPI counterstaining agent, wherein the HER-2 and TOP2A hybridization liquids comprise the set of GSP TOP2A gene probes described in Example 1.
  • the HER-2 gene and TOP2A gene detection kits are of the following four types: HER- 2 (group 1) + TOP2A (group 1) combination, HER-2 (group 2) + TOP2A (group 2) combination, HER-2 (group 1) + TOP2A (group 2) combination, HER-2 ( Group 2) + TOP2A (Group 1) combination, CSP17 probe (Chromosome 17 identification Needle, purchased from D17Z1 SpectrumAqua Probe, item number: 06J38-017), buffer component for hybridization environment (promoting hybridization), closed repetitive sequence of COT Human DNA, etc.).
  • 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 red signal indicates GSP HER-2
  • the green signal indicates GSP TOP2A
  • the cyan signal indicates CSP 17.
  • GSP TOP2A (red) and CSP17 (green) signals were counted in five clear tumor regions, and the number of GSP TOP2A and CSP 17 signals in individual nuclei was counted.
  • Example 2 Using the combination of the HER-2 gene and the TOP2A gene detection probe described in Example 1, the combination of the four detection kits described in Example 2 (HER-2 (probe group 1) + TOP2A (probe group 1), HER -2 (probe group 2) + TOP2A (probe group 2) combination, HER-2 (probe group 1) + TOP2A (probe group 2) combination, HER-2 (probe group 2) + TOP2A (Combination of probe set 1)) For 20 clinical samples (which were confirmed by pathological examination, see the table below), respectively. The detection of the two probe combinations is consistent. Compared with commercially available commercial reagents, the test results are completely consistent, and the specificity and sensitivity of the reagents are high.
  • Figure 3 and Figure 4 show the results of the combined kit detection of HER-2 (probe group 2) + TOP2A (probe group 2), as shown in Figure 3, FISH detection results of paraffin-embedded tissue samples of breast cancer, signal type The expression was 2R2G2A. The results showed that there was no abnormality in the HER-2/TOP2A gene and the chromosome 17 was normal.
  • the FISH detection results of the breast cancer tissue samples showed that the signal type was clustered R clustered G2 ⁇ 6A, and the result was judged as The HER-2/TOP2A gene was co-amplified, and chromosome 17 was multiplied.
  • the results of the combination of the other two different gene probes were the same, and the figures were omitted.
  • the detection of the HER-2 gene and the TOP2A gene can be achieved by using one probe, respectively, but the detection signal can be better by using the combination probe in combination with the probe.
  • the BAC clones of the probe set 1 and probe set 2 of the HER-2 gene of the present invention have lengths of 510 Kb and 569 Kb, respectively, and are nucleic acid mixtures containing the HER-2 gene and its both ends. .
  • the total lengths of the BAC clones of the probe sets 1 and 2 of the TOP2A gene of the present invention are: 353 Kb and 576 Kb, respectively, which are nucleic acid mixtures comprising the TOP2A gene and its both ends.
  • 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

提供一种HER-2基因和/或TOP2A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HER-2基因的BAC克隆为RP11-1021P11、RP11-62N23、RP11-1044P23，或选取BAC克隆为RP11-98J2、RP11-1065L22、CTD-2327I15；和/或选取针对TOP2A基因的BAC克隆为RP11-1152A10、CTD-3217C5，或选取BAC克隆为RP11-737D6、CTD-3087O22、RP11-48O10、CTD-2134K5；得到质粒DNA；标记。提供一种包含有HER-2基因和/或TOP2A基因检测探针的试剂盒。所述方法筛选到HER-2基因和TOP2A检测探针，信号计数行准确、快速，且结果的重复性好。

Description

HER-2基因和/或TOP2A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HER-2(ERBB2)基因和/或TOP2A基因检测探针及其制备方法和试剂盒。
背景技术
HER-2(ERBB2)基因在20％-30％的乳腺癌患者中有基因的扩增和蛋白的过度表达。HER-2是目前公认的一个乳腺癌重要的预后/预测因子，在多变量分析结果中，HER-2是肿瘤复发和总生存期长短的独立预后因素。HER-2阳性的乳腺癌浸润性强，无病生存期短，预后差。曲妥珠单抗(赫赛汀)适用于HER-2过度表达和基因扩增的的乳腺癌。TOP2A基因调节控制DNA的超螺旋状态，直接参与细胞过程，并且是蒽环类抗生素的靶酶，存在TOP2A基因异常的患者预后差，无复发生存期缩短，尤其是TOP2A基因缺失的患者预后更差。基因扩增提示肿瘤有复发的可能，或者远期的疗效下降。TOP2A基因状态在预示肿瘤化疗的反应情况和药物治疗效果中都具有一定意义。
因此正确检测和评定乳腺癌HER-2基因和TOP2A基因状态至关重要。
HER-2基因定位于17号染色体17q12区域，HER-2的异常为扩增；TOP2A基因定位于17号染色体17q21区域，TOP2A的异常情况分为扩增和缺失两种情况，FISH检测可对于这两种情况作出明确判断。
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HER-2基因和/或TOP2A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HER-2基因和/或TOP2A基因检测探针及其制备方法，所制备的探针可用于检测HER-2基因和TOP2A基因状态，即检测HER-2基因和TOP2A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HER-2基因和/或TOP2A基因检测探针的制备方法，包括以下步骤：
(1)选取针对HER-2基因的BAC克隆为RP11-1021P11、RP11-62N23、RP11-1044P23中至少一种，或选取BAC克隆为RP11-98J2、RP11-1065L22、CTD-2327I15中的至少一种；和/或选取针对TOP2A基因的BAC克隆为RP11-1152A10、CTD-3217C5中至少一种，或选取BAC克隆为RP11-737D6、CTD-3087O22、RP11-48O10、CTD-2134K5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，针对同一种基因的质粒DNA所标记的荧光素相同，针对HER-2基因和针对TOP2A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，针对TOP2A基因的所述BAC克隆为RP11-1152A10和CTD-3217C5。
在其中一个实施例中，针对TOP2A基因的所述BAC克隆为RP11-737D6、CTD-3087O22、RP11-48O10、和CTD-2134K5。
在其中一个实施例中，针对HER-2基因的BAC克隆为RP11-1021P11、RP11-62N23和RP11-1044P23。
在其中一个实施例中，针对TOP2A基因的所述BAC克隆为RP11-98J2、RP11-1065L22和CTD-2327I15。
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 PCTCN2016105718-appb-000001
FITC、Alexa
Figure PCTCN2016105718-appb-000002
Rhodamine、Texas Red、pacific
Figure PCTCN2016105718-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。
在其中一个实施例中，所述标记的温度为24℃-26℃，标记的时间为4-6小时。
本发明的另一目的是提供一种HER-2基因和TOP2A基因检测试剂盒。
实现该目的技术方案如下。
一种HER-2基因和/或TOP2A基因检测试剂盒，包括有上述HER-2基因和/或TOP2A基因检测探针。
在其中一个实施例中，包括有用于内控的17号染色体鉴别探针(CSP17)探针，该鉴别探针与HER-2基因和TOP2A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HER-2基因和/或TOP2A基因检测探针及其组合，采用FISH(Fluorescence In-Situ Hybridization)方法对HER-2基因和TOP2A基因拷贝数检测，
2.本发明优选克隆检测特异性好，灵敏度高。并通过调整标记时间和温度，限制长度探针为500bp左右，提高杂交效率和降低杂交背景。
3.信号计数行准确、快速，且结果的重复性好；通过对HER-2、TOP2A拷贝数和17号染色体同时检测和评估，有利于确定治疗方案，筛选更多受益于靶向药物的乳腺癌患者，提高患者生存率和总生存期。
4.通过本发明所述的HER-2基因和TOP2A试剂盒，从基因水平了解HER-2基因和TOP2A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，辅助乳腺癌预后判断及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HER-2基因检测探针序列的示意图。
图1B为是实施例1中TOP2A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HER-2基因和TOP2A基因检测探针FISH检测结果图。
图3为实施例4中乳腺癌组织样本FISH检测结果图，其中，检测信号类型为2R2G2A，HER-2基因未发生扩增，TOP2A基因未发生扩增。
图4为实施例4中乳腺癌组织样本FISH检测结果图，其中，检测信号类型为成簇R成簇G2A，HER-2基因和TOP2A基因发生共扩增。
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1HER-2基因和TOP2A基因检测探针的制备
一种HER-2检测探针(GSP HER-2)的制备方法，包括以下步骤：
(1)克隆筛选：挑选包含目的基因HER-2及两端序列的克隆，如图1A所示。
GSP HER-2包括两组，以下分两组检测探针分别制备。
HER-2  chr17:37,856,254-37,884,915,28,662bp
HER-2探针组1 BAC 插入片段起止位置
第一探针 RP11-1021P11 chr17：37497503…37725641(228Kb)
第二探针 RP11-62N23 chr17：37725641…37882864(157Kb)
第三探针 RP11-1044P23 chr17：37871783…38066611(195Kb)
HER-2探针组2 BAC 插入片段起止位置
第四探针 RP11-98J2 chr17：37572511…37753435(180Kb)
第五探针 RP11-1065L22 chr17：37758567…37990799(232Kb)
第六探针 CTD-2327I15 chr17：37963139…38082538(119Kb)
所述TOP2A检测探针(GSP TOP2A)的制备方法，包括以下步骤：
克隆筛选：挑选包含目的基因TOP2A及两端序列的克隆，如图1B所示。
GSP TOP2A包括两组，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。
TOP2A  chr17:38,544,773-38,574,202,29,430bp
Figure PCTCN2016105718-appb-000004
TOP2A探针组2 BAC 插入片段起止位置
第三探针 RP11-737D6 chr17：38257742…38440899(183Kb)
第四探针 CTD-3087O22 chr17：38430841…38564776(134Kb)
第五探针 RP11-48O10 chr17：38564771…38724970(160Kb)
第六探针 CTD-2134K5 chr17：38716958…38834072(117Kb)
(2)GSP HER-2和GSP TOP2A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HER-2基因或TOP2A基因的2种或3种或4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 HER-2基因的每种探针标记的荧光素为Spectrum-Orange，针对GSP TOP2A基因每种探针标记的荧光素为Spectrum-Green。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 PCTCN2016105718-appb-000005
Figure PCTCN2016105718-appb-000006
配完后震荡混匀，在25℃标记5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 TOP2A基因探针，避光、-20℃储存。
(4)GSP HER-2和GSP TOP2A基因探针验证：分别使用HER-2探针组1+TOP2A探针组1制备的杂交液，HER-2探针组2+TOP2A探针组2制备的杂交液为待检测试剂，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HER-2探针组1+TOP2A探针组1的实验结果)所示：中期染色体的FISH杂交结果图。图中可见红色信号示GSP HER-2,绿色信号示GSP TOP2A，青色信号示CSP17(用于定位17号染色体着丝粒探针,购于D17Z1SpectrumAqua Probe,货号：06J38-017)。图中可见HER-2/TOP2A/17号染色体探针信号明亮，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％；使用石蜡样本片进行杂交检测，可以清楚的记录三个靶标拷贝数。其它相应的探针验证实验结果与HER-2探针组1+TOP2A探针组2的实验结果相同，图省略。
实施例2：HER-2基因和TOP2A基因检测试剂盒制备方法
HER-2基因和TOP2A基因检测试剂盒包括有HER-2/TOP2A杂交液和DAPI复染剂两个组分，其中HER-2和TOP2A杂交液包含实施例1所述的一组GSP TOP2A基因探针和一组GSP HER-2基因探针的组合。HER-2和TOP2A基因分别有两组检测探针，两种基因的两组可以随意组合，本实施例中，所述HER-2基因和TOP2A基因检测试剂盒为四种，分别为：HER-2(组1)+TOP2A(组1)的组合、HER-2(组2)+TOP2A(组2)的组合、HER-2(组1)+TOP2A(组2)的组合、HER-2(组2)+TOP2A(组1)的组合，CSP17探针(17号染色体鉴定探 针，购于D17Z1SpectrumAqua Probe,货号：06J38-017)、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
成份 每测试
一组GSP HER-2(100ng/ul) 0.7μl
一组GSP TOP2A(100ng/ul) 0.6μl
CSP 17(50ng/ul) 0.2μl
杂交缓冲液 7μl
COT Human DNA(1mg/ml) 0.6μl
H2O 补至10μl
(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：HER-2基因和TOP2A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，红色信号示GSP HER-2，绿色信号示GSP TOP2A，青色信号示CSP 17。
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，CSP17探针(青色信号点)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40～100个肿瘤细胞核内GSP TOP2A(红色)和CSP17(绿色)信号，分别计数单个细胞核内GSP TOP2A和CSP 17的信号数。
实施例4：HER-2基因和TOP2A基因检测试剂盒临床使用评价
使用实施例1所述HER-2基因和TOP2A基因检测探针组合，实施例2所述4种检测试剂盒(HER-2(探针组1)+TOP2A(探针组1)的组合、HER-2(探针组2)+TOP2A(探针组2)的组合、HER-2(探针组1)+TOP2A(探针组2)的组合、HER-2(探针组2)+TOP2A(探针组1)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(HER-2(探针组2)+TOP2A(探针组2)的组合试剂盒检测结果，图3中所示，乳腺癌石蜡包埋组织样本FISH检测结果，信号类型表现为2R2G2A，结果判断为HER-2/TOP2A基因未见异常，17号染色体正常；图4中，乳腺癌组织样本FISH检测结果，信号类型表现为成簇R成簇G2～6A，结果判断为HER-2/TOP2A基因共扩增，17号染色体多体。其它不同两种基因探针的组合的检测结果相同，图省略。
Figure PCTCN2016105718-appb-000007
Figure PCTCN2016105718-appb-000008
HER-2(探针组1)+TOP2A(探针组2)的组合、HER-2(探针组2)+TOP2A(探针组1)的组合的检测结果，与上表所示结果相同，因篇幅的原因，具体结果省略。
本发明中，针对HER-2基因和TOP2A基因，分别各使用一种探针也能实现相应的检测，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HER-2基因的探针组1和探针组2的检测探针的BAC克隆其长度分别为：510Kb和569Kb,均为包含HER-2基因及其两端序列的核酸混合物。本发明所述针对TOP2A基因的探针组1和探针组2的检测探针的BAC克隆总长度分别为：353Kb和576Kb,均为包含TOP2A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。
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 (10)

  1. 一种HER-2基因和/或TOP2A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HER-2基因的BAC克隆为RP11-1021P11、RP11-62N23、RP11-1044P23中至少一种，或选取BAC克隆为RP11-98J2、RP11-1065L22、CTD-2327I15中的至少一种；和/或选取针对TOP2A基因的BAC克隆为RP11-1152A10、CTD-3217C5中至少一种，或选取BAC克隆为RP11-737D6、CTD-3087O22、RP11-48O10、CTD-2134K5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，针对同一种基因的质粒DNA所标记的荧光素相同，且针对HER-2基因和/或针对TOP2A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HER-2基因和/或TOP2A基因检测探针的制备方法，其特征在于，针对HER-2基因的BAC克隆为RP11-1021P11、RP11-62N23和RP11-1044P23，或为BAC克隆为RP11-98J2、RP11-1065L22和CTD-2327I15。
  3. 根据权利要求1所述HER-2基因和/或TOP2A基因检测探针的制备方法，其特征在于，针对TOP2A基因的所述BAC克隆为RP11-1152A10和CTD-3217C5，或针对TOP2A基因的所述BAC克隆为RP11-737D6、CTD-3087O22、RP11-48O10、和CTD-2134K5。
  4. 根据权利要求1所述HER-2基因和/或TOP2A基因检测探针的制备方法，其特征在于，针对HER-2基因的BAC克隆为RP11-1021P11、RP11-62N23和RP11-1044P23，以及针对TOP2A基因的所述BAC克隆为RP11-1152A10和CTD-3217C5。
  5. 根据权利要求1所述HER-2基因和/或TOP2A基因检测探针的制备方法，其特征在于，针对HER-2基因的BAC克隆为BAC克隆为RP11-98J2、RP11-1065L22和CTD-2327I15，以及针对TOP2A基因的所述BAC克隆为RP11-737D6、CTD-3087O22、RP11-48O10、和CTD-2134K5。
  6. 根据权利要求1所述HER-2基因和/或TOP2A基因检测探针的制备方法，其特征在于，所述荧光素为Alexa
    Figure PCTCN2016105718-appb-100001
    FITC、Alexa
    Figure PCTCN2016105718-appb-100002
    Rhodamine、Texas Red、pacific
    Figure PCTCN2016105718-appb-100003
    DEAC。
  7. 根据权利要求1-6任一项所述HER-2基因和/或TOP2A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24℃-26℃，时间为4-6小时。
  8. 根据权利要求1-7任一项所述的制备方法得到的HER-2基因和/或TOP2A基因检测探针。
  9. 一种HER-2基因和/或TOP2A基因检测试剂盒，其特征在于，包括有权利要求8所述的HER-2基因和/或TOP2A基因检测探针。
  10. 根据权利要求9所述HER-2基因和/或TOP2A基因检测试剂盒，其特征在于，还包括有用于内控的17号染色体鉴别探针，该鉴别探针与HER-2基因和TOP2A基因检测探针标记的荧光素的颜色不相同；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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