WO2017114004A1 - Erg基因检测探针及其制备方法和试剂盒 - Google Patents

Erg基因检测探针及其制备方法和试剂盒 Download PDF

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WO2017114004A1
WO2017114004A1 PCT/CN2016/105704 CN2016105704W WO2017114004A1 WO 2017114004 A1 WO2017114004 A1 WO 2017114004A1 CN 2016105704 W CN2016105704 W CN 2016105704W WO 2017114004 A1 WO2017114004 A1 WO 2017114004A1
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erg gene
detection
erg
probe
signal
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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 an ERG gene detection probe, a preparation method thereof and a kit.
  • Prostate cancer is a common malignant tumor disease in older men. In Europe and the United States, prostate cancer mortality ranks second among all types of malignant tumor diseases. The incidence of prostate cancer in China is much lower than that in Western countries; however, due to changes in diet, environment and lifestyle, the incidence rate has increased significantly in recent years. The staging characteristics and survival rate of prostate cancer in China have shown that the patients who are treated are mostly patients with advanced prostate cancer, and the treatment effect is not good. Therefore, early detection of prostate cancer has become an important issue.
  • Fusion of the TMPRSS2 gene to ETS is generally only present in prostate cancer, and does not occur in other tumors, benign prostatic hyperplasia, and normal epithelium. It is an early event of prostate cancer. By detecting the occurrence of this abnormality, the sensitivity and specificity of clinical detection can be improved, and a more accurate and objective diagnostic index for PSA screening and needle biopsy can be provided for the clinic.
  • the positive rate of ERG gene rearrangement in different populations and different sample groups may be related to the multivariate effects of genetic variation, disease grading, high heterogeneity of prostate cancer, sample source, and detection methods.
  • TMPRSS2-ETS can guide the administration of abiraterone.
  • the criteria for clinical pathology of prostate cancer including the Gleason score, are not sufficient to distinguish whether a patient needs immediate and aggressive treatment, resulting in overtreatment.
  • TMPRSS2-ETS fusion has unique and important clinical value as an early event in the pathogenesis of prostate cancer. It can be used as a prostate cancer diagnosis, prognosis judgment, patient stratification and treatment options by detecting patients with suspected prostate cancer and having a high family risk. Important biomarkers to compensate for the lack of other testing methods.
  • the main methods for early diagnosis of prostate cancer include serum prostate specific antigen (PSA) screening, digital rectal examination (DRE), needle biopsy and fluorescence in situ hybridization (FISH) to detect related genes.
  • PSA serum prostate specific antigen
  • DRE digital rectal examination
  • FISH fluorescence in situ hybridization
  • Serum PSA has been widely used as a screening indicator for prostate cancer, but PSA has organ specificity, not tumor specificity. Benign prostatic diseases and drug factors can also lead to elevated serum PSA.
  • DRE is the first step in the diagnosis of prostate cancer. However, sensitivity and specificity were low. Only 33% of prostate cancer diagnosed by DRE was confirmed to be an early limitation; biopsy was performed on PSA and DRE. It is suspected that a qualitative examination of prostate cancer can understand the extent of cancer tissue and pathological grade. It is the "gold standard" for prostate biopsy.
  • fusion gene detection mainly has two methods of RT-PCR and FISH. RT-PCR requires high reliability of the results of RNA degradation in high-form formaldehyde fixed and paraffin-embedded specimens.
  • FISH technology has the advantages of objective, high sensitivity (in a single cell), good accuracy (direct detection of gene status), good repeatability (standardized operation is not affected by RNA stability), non-invasive, etc., so FISH technology is used to detect prostate tissue.
  • the presence or absence of gene rearrangement of TMPRSS2 and ETS sites will be more specific.
  • Perner et al. detected 136 cases of prostate cancer by RT-PCR and FISH respectively, and found that the detection rate of TMPRSS2-ERG fusion gene in PCR was 49.2% in primary prostate cancer, and the detection rate of FISH was 60.3. %.
  • Fluorescence in situ hybridization is based on the principle of base complementation.
  • the known sequence-labeled DNA probe is hybridized with the DNA/RNA to be tested on the slide, and the hybridization signal is detected under a fluorescence microscope.
  • FISH technology links cytogenetics with changes in molecular biology, allowing tiny genetic changes to appear under the naked eye, expanding the range of cytogenetic tests and significantly improving their ability to recognize abnormal chromosomes. It has been widely used in gene amplification, translocation rearrangement and deletion detection in tumor research.
  • the foreign FISH technology is mature, the detection method is progressing rapidly, and the accuracy of detection is also continuously improved.
  • a wide variety of commercial probes, FISH detection because of its non-invasive, specific, reproducible, simple operation, etc., has gradually replaced some traditional methods in clinical, and has become a diagnostic method for certain tumors.
  • WHO recommends the use of FISH as a method for diagnosing soft tissue sarcoma and lymphoid hematopoietic tumors; FISH is currently the most accurate method for detecting HER-2 and is the gold standard for individualized treatment.
  • FISH detection reagents and related products certified by CFDA and CE which better meet the needs of clinical and scientific research.
  • One of the objects of the present invention is to provide an ERG gene detecting probe and a preparation method thereof, which can be used for detecting the state of an ERG gene, that is, detecting the rearrangement of an ERG gene, and realizing direct observation signals in cells and/or chromosomes. Types of.
  • a method for preparing an ERG gene detection probe comprising the steps of:
  • one of the set of BAC clones is RP11-720N21 and CTD-2556C4.
  • one of the set of BAC clones is RP11-360N24 and RP11-110N12.
  • the labeled fluorescein selects a fluorescent dye known in the art, preferably fluorescein is Alexa FITC, Alexa Rhodamine, Texas Red, pacific DEAC.
  • the labeling of the gene probe can be performed by labeling the corresponding fluorescein to the double-stranded nucleic acid by methods in the prior art, including but not limited to: random primer method, nick translation, etc., marker gene probe
  • the needle can be a commercially available nick translation labeling kit and/or a random primer labeling kit, preferably abbott and/or Roche's Nick Translation Kit.
  • the plasmid DNA is preferably subjected to fluorescein labeling by a random primer method or a nick translation method.
  • Another object of the present invention is to provide an ERG gene detecting kit.
  • An ERG gene detection kit comprising the above ERG gene detection probe.
  • the present invention detects the ERG gene rearrangement by FISH (Fluorescence In-Situ Hybridization) by screening the optimal ERG gene rearrangement detection probes and their combinations, and the signal counting is accurate and rapid, and the results are repeated. It is good; it supplements the lack of imported detection reagents in the clinic, which is beneficial to the subsequent application of the detection probe in clinical research, early diagnosis and treatment options for prostate cancer.
  • FISH Fluorescence In-Situ Hybridization
  • the ERG gene rearrangement detection kit of the present invention by prolonging the preferred number/length of clones, that is, increasing the signal brightness, and eliminating the influence on the detection accuracy, the ERG gene rearrangement state changes are known from the gene level, and more
  • the signal types show the genetic diversity of tumor cells in solid tissues, which can be applied in the fields of tumor biology and cytogenetics. It can help comprehensively evaluate each molecular marker and assist in the selection of clinical targeted therapy drugs and treatment options.
  • Figure 1 is a schematic illustration of the detection probe sequence in Example 1.
  • Fig. 2 is a graph showing the results of FISH detection of human peripheral blood cultured cell sheets in Example 1.
  • Example 3 is a diagram showing the results of FISH detection of prostate tissue samples in Example 4, wherein the detection signal type is 2F, and the ERG rearrangement detection is negative.
  • Example 4 is a diagram showing the results of FISH detection of a prostate tissue sample in Example 4, wherein the detection signal type is 1G1F, and the ERG rearrangement detection is positive. .
  • the GSP ERG consists of two sets of probes:
  • the first set of probes included a first probe and a second probe, as specifically listed below, which were purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • the second set of probes included a third probe and a fourth probe, as specifically listed below, which were purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • the plasmid DNA mixture was fluorescently labeled by a nick translation method, one set of probe-labeled fluorescein was Spectrum-Orange and the other set of probes labeled 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:
  • GSP ERG gene probe verification Two sets of probes were used, and the samples to be tested were subjected to human normal divisional metaphase lymphocyte droplets for two sets of probe verification (the detection method refers to the prior art and Example 3). Including medium or interim Chromosomal DNA, when fluorescent in situ hybridization, chromosomal DNA appears as a morphologically recognizable chromosome or nucleus. As shown in Figure 2: FISH hybridization results of metaphase chromosomes. The corresponding positions of the chromosomes can be seen to show red fluorescence and green fluorescence signals.
  • the ERG gene rearrangement detection kit comprises two components of a hybridization solution and a DAPI counterstain, wherein the hybridization solution comprises the two sets of GSP ERG gene probes described in Example 1 (two-color) for use 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 relevant fluorescence and DAPI need to be observed with a suitable filter block.
  • the red signal and the green signal are adjacent or fused (the red-green signal overlaps under the double-pass filter block and the yellow fused signal or the red-green signal is considered to be a single fused signal when the adjacent distance is smaller than the single signal diameter);
  • At least one set of red signal and green signal are separated by more than one or more signal diameters, red and green signals are split, such as 1F1R1G; at least one set of red-green fusion signal and one or more green signals, red signal missing, such as 1FnG ( N ⁇ 1).
  • 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 .
  • Example 2 Using the two sets of detection probes described in Example 1, the test kit described in Example 2 was performed on 20 clinical samples (formalin-fixed paraffin-embedded tissue samples, which were confirmed by pathological examination, see the table below). Detection. According to the detection method of Example 3, the detection was repeated 3 times, the results were consistent, and the repeatability of the detection results was good; the detection consistency of the two probe combinations was good.
  • Figure 3 shows that the detection signal type is 2F and the ERG rearrangement test is negative.
  • Figure 4 shows that the detection signal type is 1G1F and the ERG rearrangement test is positive.
  • 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.
  • one of the two sets of probes of the ERG gene can also be used to detect the corresponding ones, and the specific results are omitted, but the use of the combined probes can better detect the combined probes. .
  • the total length of the BAC clones of the detection probes of Group 1 and Group 2 for the ERG gene of the present invention are: 325 Kb and 266 Kb, respectively, which are nucleic acid mixtures comprising sequences at both ends of the ERG gene cleavage site.

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Abstract

提供了ERG基因检测探针及其制备方法，该方法包括以下步骤：选取两组BAC克隆，其中一组为RP11-720N21和CTD-2556C4中至少一种，和另一组为RP11-360N24和RP11-110N12中的至少一种；对克隆分别提取质粒提取，得到质粒DNA，定量；用荧光素标记。还提供了包含有ERG基因检测探针的试剂盒。

Description

ERG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ERG基因检测探针及其制备方法和试剂盒。
背景技术
前列腺癌是老年男性常见的一种恶性肿瘤性疾病。在欧美国家，前列腺癌死亡率在各类恶性肿瘤疾病中居于第二位。我国前列腺癌的发病率远低于西方国家；但由于饮食、环境和生活习惯等因素的改变，近年来发病率显著上升趋势。国内前列腺癌的分期特点和生存率共同显示，就诊患者多为晚期的前列腺癌患者，治疗效果不佳。因此，在早期检测前列腺癌已成为一项重要的课题。
TMPRSS2基因与ETS融合一般仅存在于前列腺癌中，其它肿瘤、良性前列腺增生和正常上皮中不出现。它是前列腺癌发生的早期事件，通过检测该异常发生，可以提高临床检测的灵敏度和特异性，为临床提供相比PSA筛查和穿刺活检更为准确、客观的诊断指标。但检查中需注意，ERG基因重排在不同种群、不同样本群中阳性率有差异，可能与基因变异的多因素影响、疾病分级、前列腺癌的高度异质性、样本来源、检测方法相关。
前列腺癌的发病年龄往往在65岁以上，因为年龄原因，很多病人的死亡原因并非前列腺癌，而是并发其他疾病造成死亡。因此，手术治疗并非适合于所有病人。学术界对于TMPRSS2-ETS融合基因在前列腺癌中的预后意义存在争议，多数认为与预后不良相关，研究提示缺失较断裂生存更差、复发时间更短；重排与低Gleason评分相关，拷贝数增加与高级别或晚期肿瘤相关；研究各病灶间ERG重排类型(缺失、断裂)差异可作为独立的预测转移指标。
TMPRSS2-ETS可指导阿比特龙(abiraterone)用药。简言之，前列腺癌的临床病理的标准，包括Gleason评分不足以区分患者是否需要立即和积极治疗，从而导致过度治疗。TMPRSS2-ETS融合作为前列腺癌发病的早期事件具有独特和重要的临床价值，通过对疑似前列腺癌患者、具家族史高危人群进行检测，可以作为前列腺癌诊断、预后判断、患者分层和治疗方案选择的重要生物标志物，弥补其它检测方法的不足。
用于前列腺癌早期诊断的主要方法有血清前列腺特异性抗原(PSA)筛查、直肠指诊(DRE)、穿刺活检及荧光原位杂交法(FISH)检测相关基因等。血清PSA检测已作为前列腺癌筛选指标广泛应用于临床，但PSA具有器官特异性，而非肿瘤特异性，前列腺良性疾病和药物因素也可导致血清PSA升高；DRE是诊断前列腺癌的首要步骤，但灵敏度和特异性低，DRE发现的前列腺癌只有33％被证实为早期局限性；穿刺活检是对PSA和DRE测 定后怀疑为前列腺癌的一种定性检查，可以了解癌组织范围和病理分级等，是前列腺系统穿刺活检术的“金标准”，有较高的特异性，但同时也存在出血、发热和脓肿等并发症。Tomlins等利用癌基因异常特征分析技术，证实在前列腺癌中发现跨膜丝氨酸蛋白酶编码基因TMPRSS2与ETS转录因子家族成员ERG、ETV1和ETV4之间发生融合，而良性前列腺增生标本中并检测不到该融合基因，因此具有很高的特异性。目前融合基因检测主要有RT-PCR和FISH两者方法。RT-PCR对样本要求较高部分甲醛固定、石蜡包埋标本因RNA降解影响结果的可靠性，不能直接显示标本组织形态学的关系；灵敏度高，易造成假阳性。而FISH技术具有客观、灵敏度高(单个细胞内)、准确性好(直接检测基因状态)、重复性好(标准化操作不受RNA稳定性影响)、无创等优点，因此应用FISH技术检测前列腺组织中是否存在TMPRSS2和ETS位点的基因重排将具有更高的特异性。Perner等对136例前列腺癌病例分别用RT-PCR以及FISH方法进行检测，发现在原发性前列腺癌中PCR检测TMPRSS2-ERG融合基因的检出率为49.2％，而FISH的检出率为60.3％。
荧光原位杂交技术基于碱基互补原则，用已知序列标记DNA探针与载玻片上的待测DNA/RNA互补杂交，在荧光显微镜下检测杂交信号判断结果。FISH技术将细胞遗传学和分子生物学改变联系起来，让微小的基因改变显现于肉眼之下，拓展了细胞遗传学检测的范围，显著提高了其识别异常染色体的能力。目前已被广泛应用于肿瘤研究中的基因扩增、易位重排及缺失等检测。
国外FISH技术成熟，检测方法进展很快，检测的精确率也不断提高。各种商业化的探针种类繁多，FISH检测因其无创、特异、重复性高、操作简单等优点，在临床上已经逐渐取代一些传统方法，成为某些肿瘤的诊断方法。如WHO推荐使用FISH技术作为诊断软组织肉瘤和淋巴造血系统肿瘤的方法；FISH检测是目前公认的最为准确的HER-2检测方法，是个体化治疗的金标准。目前，CFDA和CE认证的FISH检测试剂和相关产品极多，较好的满足了临床和科研的需要。国内FISH技术相对于国外起步较晚，应用主要集中在基础医学研究领域。不过，通过这一阶段的发展，已经为临床检测积累了大量科学数据和试验方法，技术上益趋成熟，也出现了基于该技术的检测试剂，但种类少，质量相对于国外试剂也有较大的差距。这些问题对于满足国内市场需求和推广临床应用极为不利。
发明内容
本发明的目的之一是提供一种ERG基因检测探针及其制备方法，所制备的探针可用于检测ERG基因状态，即ERG基因重排的检测，实现细胞和/或染色体中直接观察信号类型。
实现上述目的的技术方案如下。
一种ERG基因检测探针的制备方法，包括以下步骤：
(1)选取两组BAC克隆，其中一组为RP11-720N21和CTD-2556C4中至少一种，另一组为RP11-360N24和RP11-110N12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，两组质粒DNA所标记的荧光素不相同，同组质粒DNA所标记的荧光素相同，即得。
在其中一个实施例中，其中一组所述BAC克隆为RP11-720N21和CTD-2556C4。
在其中一个实施例中，其中一组所述BAC克隆为RP11-360N24和RP11-110N12。
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 PCTCN2016105704-appb-000001
FITC、Alexa
Figure PCTCN2016105704-appb-000002
Rhodamine、Texas Red、pacific
Figure PCTCN2016105704-appb-000003
DEAC。
在其中一个实施例中，基因探针的标记可以采用现有技术中的方法将相应荧光素标记至双链核酸上，所述方法包括但不限于：随机引物法、切口平移等，标记基因探针可以使用市售的缺口平移标记试剂盒和/或随机引物标记试剂盒，优选abbott和/或Roche公司的Nick Translation Kit。本发明步骤(3)优选采用随机引物法、切口平移法对质粒DNA进行荧光素标记。
本发明的另一目的是提供一种ERG基因检测试剂盒。
实现该目的技术方案如下。
一种ERG基因检测试剂盒，包括有上述ERG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ERG基因重排检测探针及其组合，采用FISH(Fluorescence In-Situ Hybridization)方法对ERG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，进行前列腺癌早期诊断、治疗选择等方面。
2、通过本发明所述的ERG基因重排检测试剂盒，通过延长优选克隆数量/长度，即提高信号亮度，又消除对检测准确性的影响，从基因水平了解ERG基因重排状态改变，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1为是实施例1中检测探针序列的示意图。
图2是实施例1中人外周血培养细胞片FISH检测结果图。
图3为实施例4中前列腺组织样本FISH检测结果图，其中，检测信号类型为2F，ERG重排检测阴性。
图4为实施例4中前列腺组织样本FISH检测结果图，其中，检测信号类型为1G1F，ERG重排检测阳性。。
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1  ERG基因检测探针的制备
本实施所述ERG检测探针的制备方法，包括以下步骤：
(1)挑选包含目的基因ERG及两端序列的克隆，如图1所示。GSP ERG包括两组探针：
第一组探针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。
第二组探针包括第三探针和第四探针，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。
第一组探针
  BAC 插入片段起止位置
第一探针 RP11-720N21 chr21:39436974…39647010(210Kb)
第二探针 CTD-2556C4 chr21:39624588…39761785(137Kb)
第二组探针
   BAC 插入片段起止位置
第三探针 RP11-360N24 chr21:40013910…40216958(203Kb)
第四探针 RP11-110N12 chr21:40133807…40280238(146Kb)
(2)GSP ERG基因检测探针制备：使用Qiagen公司的Plasmid Maxi Kit，按照说明书要求的操作方法对不同BAC克隆分别进行超低拷贝质粒DNA提取，通过测定260nm和280nm处的吸光度对质粒DNA定量；采用高压灭菌的超纯水稀释为200ng/ul,采用1.5ml的离心管分装，最后将分别得到的2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，一组探针标记的荧光素为Spectrum-Orange，另一组探针标记为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 PCTCN2016105704-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，要求在50bp～500bp之间存在弥散的带。
对标记产物进行乙醇沉淀和浓缩，按如下方案在1.5ml离心管中依次加入醋酸钠和无水乙醇，避光、冰上配制：
标记产物            45ul
醋酸钠(3mol/L)      5ul
无水乙醇            125ul
混匀后置于-70℃冰箱中至少2小时，4℃13000rpm离心30分钟，小心去上清，勿搅动沉淀，加入1ml的70％乙醇，4℃13000转/分钟离心15分钟，小心去上清，勿搅动沉淀，避光干燥。使用1ul纯化水溶解沉淀，获得GSP ERG基因探针，避光、-20℃储存。
(4)GSP ERG基因探针验证：分别使用两组探针，使用人类正常分裂中期淋巴细胞滴片进行待测样本进行两组探针验证(检测方法参考现有技术和实施例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2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。
实施例2：ERG基因检测试剂盒制备方法
ERG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所述的两组GSP ERG基因探针(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：
(1)杂交液配制
Figure PCTCN2016105704-appb-000005
(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：ERG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，将其放入室温二甲苯(或环保脱蜡剂)中10分钟；
1.3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；
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(或环保脱蜡剂)；
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，再将其放入灭菌纯化水中室温洗涤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)；
1.8冷却至室温后，取出玻片，自来水冲洗干净；
1.9取出玻片，将其放入室温2×SSC中3分钟；
1.10取出玻片，室温晾干；
1.11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；
1.12将多余液体甩去，将其放入室温2×SSC中5分钟；
1.13取出玻片，再将其放入另一缸室温2×SSC中5分钟；
1.14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；
1.15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。
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：红色信号和绿色信号相邻或融合(双通滤块下观察红绿信号重叠呈黄色融合信号或红绿信号相邻距离小于单个信号直径时认为单个融合信号)；
6.2异常信号：至少有一组红色信号和绿色信号相距大于一个或更多信号直径，红绿信号分裂，如1F1R1G；至少有一组红绿融合信号和一个及以上绿色信号，红色信号缺失，如1FnG(n≥1)。
7.设定阴性阈值
随机选取10例以上的前列腺增生样本当阴性质控片。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：ERG基因检测试剂盒临床使用评价
使用实施例1所述两组检测探针，实施例2所述检测试剂盒对20份临床样本(福尔马林固定石蜡包埋组织样本，其经过病理检测确诊，具体见下表)，进行检测。根据实施例3的检测方法重复检测3次，结果相符，检测结果的重复性好；两种探针组合的检测一致性佳。图3为检测信号类型为2F，ERG重排检测阴性。图4为检测信号类型为1G1F，ERG重排检测阳性。
Figure PCTCN2016105704-appb-000006
Figure PCTCN2016105704-appb-000007
从检测结果可知，在对这些样本进行分子标志物检测后，可以据检测结果对样本进行分子分型，依据检测指标的意义，用于临床治疗方案制定、用药选择和疗效判断。
本发明中，分别各使用ERG基因两组探针中的一种探针也能实现相应的检测，具体结果省略，但相对探针组合使用而言，组合探针的使用，检测信号会更好。理论上探针长度越长，实际检测时获得的荧光信号亮度越明亮，但因为可能涉及到更多基因序列，所得到的信号复杂性可能性增多，对检测实现的难度也增强。本发明所述针对ERG基因的组1和组2的检测探针的BAC克隆总长度分别为：325Kb和266Kb,为包含ERG基因断裂位点两端序列的核酸混合物。
发明人在对本发明所述探针验证中发现，较长的检测探针确实获得更强的荧光信号，并且在对临床样本的检测验证中也获得了相同的结果。因此，在荧光探针的设计中，可以通过适当延长荧光探针长度增加信号亮度，但具体如何组合使用，存在的一定的技术困难，要实现很好的检测结果，除了设计中的经验之外，还需通过临床样本验证评估信号类型差异，需要发明人付出大量艰辛的工作，寻求合适的探针组合。
所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (6)

  1. 一种ERG基因检测探针的制备方法，其特征在于，包括以下步骤：
    (1)选取两组BAC克隆，其中一组为RP11-720N21和CTD-2556C4中至少一种，和另一组为RP11-360N24和RP11-110N12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，两组质粒DNA所标记的荧光素不相同，同组质粒DNA所标记的荧光素相同，即得。
  2. 根据权利要求1所述的ERG基因检测探针的制备方法，其特征在于，其中一组所述BAC克隆为RP11-720N21和CTD-2556C4。
  3. 根据权利要求1所述的ERG基因检测探针的制备方法，其特征在于，其中一组所述BAC克隆为RP11-360N24和RP11-110N12。
  4. 根据权利要求1-3任一项所述的ERG基因检测探针的制备方法，其特征在于，所述荧光素为Alexa
    Figure PCTCN2016105704-appb-100001
    FITC、Alexa
    Figure PCTCN2016105704-appb-100002
    Rhodamine、Texas Red、pacific
    Figure PCTCN2016105704-appb-100003
    DEAC。
  5. 根据权利要求1-4任一项所述的制备方法得到的ERG基因检测探针。
  6. 根据权利要求5所述ERG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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