技术领域technical field
本发明涉及的是一种分子生物技术领域的药物敏感度检测方法，包括引物、探针及其试剂盒，具体是一种用于他莫西芬抗肿瘤药物敏感度检测的方法，针对他莫西芬药物使用者基因内3个生物标记的引物、探针及其试剂盒。The present invention relates to a method for detecting drug sensitivity in the field of molecular biology technology, including primers, probes and kits thereof, specifically a method for detecting the sensitivity of tamoxifen antineoplastic drugs, targeting tamoxifen Primers, probes and kits for three biomarkers in the Sifen drug user gene.
背景技术Background technique
化疗在目前是恶性肿瘤治疗的重要手段，临床化疗的效果对于肿瘤患者的治疗和提高生存期有着至关重要的影响。对于不同的患者，不同的化疗药物的效果差异很大。肿瘤患者对化疗的反应不同，很多时候是由于对化疗药物敏感性存在差异。由于临床医生往往只能凭经验对不同患者使用某种化疗药物或化疗方案，带有一定的盲目性，因此很多肿瘤患者的化疗效果并不理想，往往达不到化疗的预期目的。所以，一个能够在化疗前筛选出针对性强、患者敏感度较高化疗药物的检测方法，对于提高个体化治疗效果，提高化疗的成功率，有着重要的意义。Chemotherapy is currently an important means of treatment of malignant tumors, and the effect of clinical chemotherapy has a crucial impact on the treatment of cancer patients and the improvement of survival. The effects of different chemotherapy drugs vary widely from patient to patient. Cancer patients respond differently to chemotherapy, often due to differences in sensitivity to chemotherapy drugs. Because clinicians often can only use certain chemotherapy drugs or chemotherapy regimens for different patients based on experience, with a certain degree of blindness, the chemotherapy effect of many cancer patients is not ideal, and the expected purpose of chemotherapy is often not achieved. Therefore, a detection method that can screen out highly targeted and highly sensitive chemotherapy drugs before chemotherapy is of great significance for improving the effect of individualized treatment and the success rate of chemotherapy.
近年来国内外肿瘤和化疗药物研究领域相继创建了一系列体内或体外预测肿瘤化疗药物敏感性的方法。常用的有裸鼠皮下移植药敏测定法、人体肿瘤细胞集落测定法、放射性标记代谢物前体掺入法、MTT比色、三磷酸腺苷法和快速荧光分析等等。这些方法均存在一些缺点，要么费用昂贵且检测周期长，不适合常规使用；要么需要在体外培养原发肿瘤，检测成功率低。这些缺陷把肿瘤化疗药敏实验限制在了研究性的实验室里，很少有大规模使用的报道。In recent years, a series of in vivo or in vitro methods for predicting the sensitivity of tumor chemotherapy drugs have been created in the field of tumor and chemotherapy drug research at home and abroad. Commonly used nude mouse subcutaneous transplantation drug susceptibility assay, human tumor cell colony assay, radiolabeled metabolite precursor incorporation method, MTT colorimetry, adenosine triphosphate method and rapid fluorescence analysis, etc. These methods all have some disadvantages, either they are expensive and have a long detection period, and are not suitable for routine use; or they need to culture primary tumors in vitro, and the detection success rate is low. These shortcomings limit the drug sensitivity test of tumor chemotherapy to research laboratories, and there are few reports of large-scale use.
药物基因组学（pharmacogenomics）研究的进展为从分子水平研制和开发新一代的，针对药物敏感性进行检测的方法提供了理论基础和大量的实验数据。实验证明，个体基因水平上的差异，包括基因多态性、基因突变和表达差异等，与肿瘤患者对药物的敏感性密切相关。通过检测患者在这些基因上与药物敏感性相关的生物标记，可以预测患者对肿瘤化疗药物的敏感性，帮助进一步指导选择合理的化疗方案。我们通过设计试剂盒，检测人类基因上的多个SNP生物标记，可以方便、快速的预测他莫西芬药物对患者个体的疗效。The progress of pharmacogenomics (pharmacogenomics) research provides a theoretical basis and a large amount of experimental data for the research and development of a new generation of methods for drug sensitivity detection from the molecular level. Experiments have shown that differences at the individual gene level, including gene polymorphisms, gene mutations, and expression differences, are closely related to the sensitivity of cancer patients to drugs. By detecting the biomarkers related to drug sensitivity in these genes, the sensitivity of patients to chemotherapy drugs can be predicted, which can help to further guide the selection of reasonable chemotherapy regimens. By designing a kit to detect multiple SNP biomarkers on human genes, we can conveniently and quickly predict the curative effect of tamoxifen on individual patients.
发明内容Contents of the invention
本发明的目的是利用SNP标记检测方便快捷，准确率高的优点，采用Seqnome检测技术，提供一种用于他莫西芬抗肿瘤药物敏感度检测的方法，包括PRC引物、延伸引物和试剂盒。The purpose of the present invention is to utilize the advantages of SNP marker detection to be convenient and quick, and the accuracy rate is high, adopt Seqnome detection technology, provide a kind of method that is used for the detection of tamoxifen antineoplastic drug sensitivity, comprise PRC primer, extension primer and test kit .
本发明涉及一种用于他莫西芬抗肿瘤药物敏感度检测的PCR扩增引物，含有SEQIDNo.1~3所示的上游引物和SEQIDNo.4~6所示的下游引物。The invention relates to a PCR amplification primer used for detecting the sensitivity of tamoxifen antineoplastic drugs, which contains upstream primers shown in SEQ ID No. 1-3 and downstream primers shown in SEQ ID No. 4-6.
本发明涉及一种用于他莫西芬抗肿瘤药物敏感度检测的单碱基延伸引物，含有SEQIDNo.7~9所示的单碱基延伸引物。The invention relates to a single-base extension primer used for detecting the sensitivity of tamoxifen antitumor drugs, which comprises the single-base extension primers shown in SEQ ID Nos. 7-9.
本发明设计一种试剂盒，由PCR扩增试剂组、SAP酶试剂组和iPLEX试剂组构成，其中PCR扩增试剂组含有：PCR缓冲液、MgCl2、dNTP混合液、HotstarTaq酶、纯水和如SEQIDNo.1~3所示的上游引物和如SEQIDNo.4~6所示的下游引物；SAP酶试剂组含有：SAP缓冲液、SAP酶和纯水；iPLEX试剂组含有：iPLEX缓冲液、ddNTP混合液、iPLEX聚合酶、纯水和如SEQIDNo.7~9所示的单碱基延伸引物。The present invention designs a kit, which is composed of PCR amplification reagent group, SAP enzyme reagent group and iPLEX reagent group, wherein the PCR amplification reagent group contains: PCR buffer, MgCl2 , dNTP mixed solution, HotstarTaq enzyme, pure water and Upstream primers as shown in SEQIDNo.1~3 and downstream primers as shown in SEQIDNo.4~6; SAP enzyme reagent set contains: SAP buffer, SAP enzyme and pure water; iPLEX reagent set contains: iPLEX buffer, ddNTP Mixture, iPLEX polymerase, pure water and single base extension primers as shown in SEQ ID No.7-9.
上述标记组合优选利用飞行质谱（MALDI-TOF）方法进行检测，检测方法包括以下步骤（1）提取基因组DNA，包括提取抗凝血DNA和口腔上皮细胞DNA；（2）飞行质谱检测SNP多态；（3）根据SNP分型结果推断他莫西芬药物敏感度。The above marker combination is preferably detected by mass spectrometry of flight (MALDI-TOF), and the detection method includes the following steps (1) extracting genomic DNA, including extracting anticoagulant DNA and oral epithelial cell DNA; (2) detecting SNP polymorphisms by mass spectrometry of flight; (3) The drug sensitivity of tamoxifen was inferred based on the SNP typing results.
本发明对ABCB1基因上的三个SNP生物标记进行分型，建立了一个快速，简便，且成本低廉的检测试剂盒，该试剂盒准确度高，灵敏性好，具有很强的实用价值。The invention performs typing on three SNP biomarkers on the ABCB1 gene, and establishes a fast, simple and low-cost detection kit. The kit has high accuracy, good sensitivity and strong practical value.
实施例Example
以下实施例，对本发明的具体实施方式进行详细描述，实施例用于说明本发明，但不用来限制本发明的范围，实施例1：采用飞行质谱方法对肿瘤患者进行SNP分型及他莫西芬药物敏感度检测方法：The following examples describe the specific implementation of the present invention in detail. The examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Example 1: Carrying out SNP typing and tamoxicil on tumor patients by using flight mass spectrometry Fen drug sensitivity detection method:
一）提取基因组DNA：本试验的样本是使用刮棒采集的口腔粘膜细胞，室温保存。刮棒采样DNA提取的具体步骤如下：1) Genomic DNA extraction: The samples in this test are oral mucosal cells collected with a scraper and stored at room temperature. The specific steps of DNA extraction by scraping stick sampling are as follows:
1）将口腔刮棒上的脱落细胞溶于400μL1X裂解液中，加入1μL蛋白酶K，56度水浴30分钟；1) Dissolve the exfoliated cells on the oral scraper in 400 μL of 1X lysate, add 1 μL of proteinase K, and bathe in 56-degree water for 30 minutes;
2）取出样品置冰水浴1分钟，加入6mol/LNaCl150μL，剧烈振荡15-20秒，13000转离心10分钟；2) Take out the sample and place it in an ice-water bath for 1 minute, add 150 μL of 6mol/L NaCl, shake vigorously for 15-20 seconds, and centrifuge at 13,000 rpm for 10 minutes;
3）吸取上清液至新的离心管中，加入1.1mL无水乙醇，上下颠倒10次至絮状DNA沉淀出现。室温放置10分钟，13000转离心2分钟沉淀DNA；3) Pipette the supernatant into a new centrifuge tube, add 1.1mL absolute ethanol, invert up and down 10 times until flocculent DNA precipitates appear. Place at room temperature for 10 minutes, centrifuge at 13,000 rpm for 2 minutes to precipitate DNA;
4）弃去上清液，再加入0.25mL70%乙醇洗涤DNA沉淀，室温放置1分钟后，13000转离心5分钟；4) Discard the supernatant, then add 0.25mL of 70% ethanol to wash the DNA pellet, leave it at room temperature for 1 minute, and then centrifuge at 13000 rpm for 5 minutes;
5）用移液器小心吸取乙醇，然后放入通风橱中通风10分钟，然后用35μLTE溶解DNA；6）DNA可在4度保存1-2个月，或者存放在-20度长期保存；5) Carefully draw ethanol with a pipette, then put it in a fume hood for 10 minutes, and then dissolve DNA with 35 μLTE; 6) DNA can be stored at 4 degrees for 1-2 months, or stored at -20 degrees for long-term storage;
7）使用18μLPCRMasterMix反应液以及2μLDNA模板，PCR扩增β-actin片断，1.5%琼脂糖凝胶，120V电泳15分钟，观察结果合格后转移至PCR管中；7) Use 18 μL of PCRMasterMix reaction solution and 2 μL of DNA template to amplify the β-actin fragment by PCR, electrophoresis at 120V for 15 minutes on 1.5% agarose gel, and transfer to a PCR tube after passing the observation result;
8）紫外分光光度计SMA3000测定溶液中DNA的浓度和A260/A280比值，测量3次取平均值，浓度应在30ng/μL，A260/A280比值应该在1.7-2.0之间，合格的DNA4℃取用或-20℃保存；8) Measure the DNA concentration and A260/A280 ratio in the solution with an ultraviolet spectrophotometer SMA3000, take the average value of 3 measurements, the concentration should be 30ng/μL, and the A260/A280 ratio should be between 1.7-2.0. Use or store at -20°C;
二）飞行质谱检测SNP多态，采用本发明中所述试剂盒对样本中的SNP生物标记进行检测，下面为检测过程：2) Detection of SNP polymorphism by flight mass spectrometry, using the kit described in the present invention to detect the SNP biomarkers in the sample, the following is the detection process:
1）引物与DNA稀释以及质检：PCR引物稀释至终浓度为0.5μM；根据Sequenom的稀释公式，按照单碱基延伸引物的分子量将引物稀释至终浓度为7-14μM之间；1) Primer and DNA dilution and quality inspection: PCR primers were diluted to a final concentration of 0.5 μM; according to Sequenom’s dilution formula, primers were diluted to a final concentration of 7-14 μM according to the molecular weight of the single-base extension primer;
2）PCR扩增：在每个384孔PCR板中加入1μLDNA和4μLPCR扩增试剂，共5μL，按照PCR仪程序1进行扩增：2) PCR amplification: Add 1 μL of DNA and 4 μL of PCR amplification reagent to each 384-well PCR plate, totaling 5 μL, and perform amplification according to PCR instrument program 1:
PCR程序1：94℃15minPCR program 1: 15min at 94°C
94℃20sec 94℃20sec
56℃30sec45cycles56℃30sec45cycles
72℃1min72℃1min
72℃3min72℃3min
4℃∞4°C∞
PCR扩增结束后，将384孔板短暂离心后备用，可在4℃保存；After PCR amplification, centrifuge the 384-well plate briefly and store it at 4°C;
3）SAP酶处理：在PCR扩增后的产物加入2μL的SAP酶试剂，共7μL。按照PCR仪程序2进行扩增：3) SAP enzyme treatment: Add 2 μL of SAP enzyme reagent to the PCR-amplified product, a total of 7 μL. Amplify according to PCR instrument program 2:
PCR程序2：37℃，40min85℃，5min4℃，∞ PCR program 2: 37°C, 40min, 85°C, 5min, 4°C, ∞
SAP酶处理结束，将384孔板取出短暂离心备用；After the SAP enzyme treatment is over, take out the 384-well plate and centrifuge briefly;
4）iPLEX延伸：在SAP酶处理后的产物加入2μL的iPLEX试剂，共9μL。按照PCR仪程序3进行扩增；4) iPLEX extension: add 2 μL of iPLEX reagent to the product after SAP enzyme treatment, a total of 9 μL. Amplify according to PCR instrument program 3;
PCR程序3：94℃30secPCR program 3: 94°C 30sec
94℃5sec 94℃5sec
52℃5sec5cycle40cycle 52℃5sec5cycle40cycle
80℃5sec80℃5sec
72℃3min72℃3min
4℃∞4°C∞
iPLEX延伸结束，将384孔板取出短暂离心备用；After the iPLEX extension is over, take out the 384-well plate and centrifuge briefly;
5）树脂纯化与数据收集5) Resin purification and data collection
将反应产物（共9μL）加入25μL水进行稀释，使用树脂进行脱盐；将脱盐处理后的样品点在芯片靶点上，自然结晶，上机进行质谱检测，并收集数据；The reaction product (9 μL in total) was diluted with 25 μL of water, and desalted with resin; the desalted sample was placed on the chip target, naturally crystallized, and the machine was used for mass spectrometry detection and data collection;
三）根据SNP分型结果推断他莫西芬药物敏感度：3) Deduce tamoxifen drug sensitivity based on SNP typing results:
根据肿瘤患者样品DNA中3个SNP生物标记的分型结果，我们对患者他莫西芬药物敏感度进行评价，如果样品DNA中含有对他莫西芬药物耐药的基因型，我们将对患者进行风险提示。According to the typing results of the 3 SNP biomarkers in the DNA of tumor patient samples, we will evaluate the drug sensitivity of the patient to tamoxifen. If the sample DNA contains a genotype resistant to tamoxifen, we will Give risk warning.
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| EP0711870A1 (en)* | 1991-11-01 | 1996-05-15 | The Procter & Gamble Company | Soft absorbent tissue paper comprising a biodegradable quaternized di-methylated amine-ester compound and a temporary wet strength resin |
| CN101463395A (en)* | 2008-12-26 | 2009-06-24 | 中国农业科学院兰州兽医研究所 | Reagent kit for detecting porcine reproductive and respiratory syndrome virus and detecting method |
| CN103045734A (en)* | 2012-12-19 | 2013-04-17 | 上海迪道科技有限公司 | Tamoxifen anticancer drug sensitivity detection method |
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0711870A1 (en)* | 1991-11-01 | 1996-05-15 | The Procter & Gamble Company | Soft absorbent tissue paper comprising a biodegradable quaternized di-methylated amine-ester compound and a temporary wet strength resin |
| CN101463395A (en)* | 2008-12-26 | 2009-06-24 | 中国农业科学院兰州兽医研究所 | Reagent kit for detecting porcine reproductive and respiratory syndrome virus and detecting method |
| CN103045734A (en)* | 2012-12-19 | 2013-04-17 | 上海迪道科技有限公司 | Tamoxifen anticancer drug sensitivity detection method |
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