CN103966343A - Fgfr1基因实时pcr检测方法及其试剂盒和引物 - Google Patents
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Abstract
本发明涉及基因检测领域，尤其涉及FGFR1基因实时PCR检测方法及其试剂盒和引物。一种FGFR1基因实时PCR检测方法，该方法利用寡聚核苷酸引物对从肿瘤组织或细胞中提取的基因组DNA或者游离于血液或尿液中的为模板进行PCR扩增，所述的寡聚核苷酸引物的序列如下：FP 5’-GCTAGGTGCCGAGGGTGTT-3’RP 5’-ACTGCAGGCTCCTTCAGAAC-3’；上述的方法用于非疾病的诊断和治疗。本发明利用FGFR抑制剂对肿瘤病人进行个性化的靶向治疗，FGFR1基因扩增检测试剂盒的开发将带来极大的社会效益。
Description
技术领域
本发明涉及基因检测领域，尤其涉及FGFR1基因实时PCR检测方法及其试剂盒和引物。
背景技术
成纤维生长因子受体1(FGFR1)分子靶点是一种新型有效蛋白靶点，近些年发现在部分乳腺癌、膀胱癌、恶性神经胶质瘤、脑膜瘤等肿瘤组织中FGFR1基因高度扩增，同时伴有FGFR1蛋白过度表达，其肿瘤发生机理是成纤维生长因子(FGF)与FGFR1结合，从而激发受体的酪氨酸激酶活性，使结合于激酶区的底物（信号分子）发生磷酸化，引发细胞内的一系列信号传导途径，刺激细胞繁殖、肿瘤生长，FGFR1可以与多种FGF结合。含有FGFR1基因高度扩增的肿瘤细胞对FGFR1信号传导有着依赖性，因此可以应用FGFR1抑制剂来抑制这些肿瘤细胞生长、导致肿瘤细胞凋亡，从而达到治疗癌症的效果。事实上，国际上有十多家公司（包括Boehringer Ingelheim, 诺华，施贵宝等等）已经开发了十多种不同的能够抑制FGFR1的药物。
发明内容
本发明的第一个目的是提供一种FGFR1基因实时PCR检测方法，本发明的第二个目的是提供上述方法使用的试剂盒，本发明的第三个目的是提供上述的方法采用的寡聚核苷酸引物。该方法具有稳定性好，灵敏性高的特点。
为了实现上述的第一个目的，本发明采用了以下的技术方案：
一种FGFR1基因实时PCR检测方法，该方法利用寡聚核苷酸引物对从肿瘤组织或细胞中提取的基因组DNA或者游离于血液或尿液中的为模板进行PCR扩增，所述的寡聚核苷酸引物的序列如下：
FP 5’-GCTAGGTGCCGAGGGTGTT-3’
RP 5’-ACTGCAGGCTCCTTCAGAAC-3’；
上述的方法用于非疾病的诊断和治疗。
作为优选，该方法还包括使用一个荧光标记探针。再优选，所述的荧光标记探针选用TaqMan探针。最优选，所述的荧光标记探针的核苷酸顺序为5’-FAM-CAAAGGTTAGGGAGGCAC-MGB-3’。
所述肿瘤细胞或组织可以是任何人肿瘤细胞和组织。作为优选，所述的肿瘤细胞或组织为乳腺癌、非细胞肺癌、膀胱癌、口腔鳞癌、食道鳞状细胞癌、卵巢癌或前列腺癌。再优选，所述的肿瘤细胞或组织为乳腺癌或肺鳞癌细胞或组织。
为了实现上述的第二个目的，本发明采用了以下的技术方案：
一种用于FGFR1基因实时PCR检测的试剂盒，该试剂盒包括一组寡聚核苷酸引物，所述的寡聚核苷酸引物的序列如下：
FP 5’-GCTAGGTGCCGAGGGTGTT-3’
RP 5’-ACTGCAGGCTCCTTCAGAAC-3。
作为优选，该试剂盒还包括荧光标记探针。再优选，所述的荧光标记探针选用TaqMan探针。最优选，所述的荧光标记探针的核苷酸顺序为5’-FAM-CAAAGGTTAGGGAGGCAC-MGB-3’。
为了实现上述的第三个目的，本发明采用了以下的技术方案：
寡聚核苷酸引物，所述的寡聚核苷酸引物的序列如下：
FP 5’-GCTAGGTGCCGAGGGTGTT-3’
RP 5’-ACTGCAGGCTCCTTCAGAAC-3’。
本发明利用FGFR抑制剂对肿瘤病人进行个性化的靶向治疗，首先需要筛选出那些在癌细胞中有FGFR1基因扩增的病人。因此本发明涉及一种基因诊断试剂盒，通过检测肿瘤组织中FGFR1的扩增情况，确定癌症的病变因素和制定有效治疗方案。如果存在FGFR1高度扩增情况，可以推荐患者接受FGFR抑制剂药物治疗，使适宜FGFR靶向治疗的患者获益；如果不存在FGFR1高度扩增，应及时考虑其它治疗药物，避免使患者蒙受药物的潜在毒性作用及不必要的经济负担，并错过最佳治疗时机。FGFR1基因扩增检测试剂盒的开发将带来极大的社会效益。
附图说明
图1 显示本发明的扩增体系检测FGFR1：GAPDH=1:1基因扩增图。
图2显示本发明的扩增体系检测FGFR1：GAPDH=2:1基因扩增图。
图3显示本发明的扩增体系扩增 HT29细胞株基因组DNA的结果图。
图4显示本发明的扩增体系扩增 SW480细胞株基因组DNA的结果图。
具体实施方式
实施例 1
本发明以构建的质粒为模板，构建FGFR1基因实时荧光PCR扩增反应体系，以FAM为信号检测通道，根据FGFR1基因以及GAPDH持家基因序列分别设计特异性探针和双引物，通过引物和检测体系的优化实现高准确度的特异性检测。检测FGER1基因扩增的方法包括一下步骤：
（1）针对FGFR1基因和GAPDH持家基因序列，应用Premier 5.0设计特异性探针和双引物，引物和探针序列如表1所示。
表1 FGFR1及GAPDH引物及探针信息
| 名称 | FGFR1基因(F1) | GAPDH基因(G1) |
| FP | 5’-GCTAGGTGCCGAGGGTGTT-3’ | 5’- GGAAGCTCAAGGGAGATAA -3’ |
| RP | 5’-ACTGCAGGCTCCTTCAGAAC-3’ | 5’- AATGCAGCATCTCCTTACC -3’ |
| Probe | 5’-FAM-CAAAGGTTAGGGAGGCAC-MGB-3’ | 5’-FAM-AACCTCTTGGGCCCTC-MGB-3’ |
（2）质粒的提取采用TIANGEN(HighPure Plasmid Kit，DP116) 的质粒提取试剂盒进行提取，具体提取操作步骤按试剂盒说明书操作。所提DNA 溶于Tris-HCl 中(10mmol/L，PH8.0)，经紫外分光光度计检测提取质量，确定其浓度，然后将FGFR1与GAPDH质粒分别按1:1和2:1的拷贝数比例混合。用Tris-HCl(10mmol/L，PH8.0)溶液调整混合质粒浓度到105copies/μL 作为PCR 模板，取5μL 进行PCR 反应扩增。
（3）建立PCR扩增反应体系：
| 10×buffer | 1× |
| dNTP | 0.25mM |
| Mg2+ | 3mM |
| 双引物 | 200nM |
| 探针 | 100nM |
| Taq酶 | 1U |
| 模板 | 5.0ul |
| 总体积 | 25ul |
实时PCR 反应条件是95oC预变性2分钟；95oC变性20 秒，60oC退火32秒，45个循环。在退火时检测FAM 荧光信号。 检测结果如图1、图2。
实施例2
以肠癌细胞系HT29细胞株和正常人血淋巴细胞SW480细胞株的DNA为模板，TaqMan探针法检测两个体系的扩增情况。
建立PCR扩增反应体系：
| 10×buffer | 1× |
| dNTP | 0.25mM |
| Mg2+ | 3mM |
| 双引物 | 200nM |
| 探针 | 100nM |
| Taq酶 | 1U |
| 模板 | 10ng |
| 总体积 | 25ul |
实时PCR 反应条件是95oC预变性2分钟；95oC变性20 秒，60oC退火32秒，45个循环。在退火时检测FAM 荧光信号。
结果表明两个体系的扩增效率基本相等，结果如图3、图4所示。
Claims (10)
1.一种FGFR1基因实时PCR检测方法，其特征在于：该方法利用寡聚核苷酸引物对从肿瘤组织或细胞中提取的基因组DNA或者游离于血液或尿液中的为模板进行PCR扩增，所述的寡聚核苷酸引物的序列如下：
FP 5’-GCTAGGTGCCGAGGGTGTT-3’
RP 5’-ACTGCAGGCTCCTTCAGAAC-3’；
上述的方法用于非疾病的诊断和治疗。
2.根据权利要求1所述的一种FGFR1基因实时PCR检测方法，其特征在于：该方法还包括使用一个荧光标记探针。
3.根据权利要求1所述的一种FGFR1基因实时PCR检测方法，其特征在于： 荧光标记探针选用TaqMan探针。
4.根据权利要求1所述的一种FGFR1基因实时PCR检测方法，其特征在于：肿瘤细胞或组织为乳腺癌、非细胞肺癌、膀胱癌、口腔鳞癌、食道鳞状细胞癌、卵巢癌或前列腺癌。
5.根据权利要求4所述的一种FGFR1基因实时PCR检测方法，其特征在于：肿瘤细胞或组织为乳腺癌或肺鳞癌细胞或组织。
6.一种用于FGFR1基因实时PCR检测的试剂盒，其特征在于该试剂盒包括一组寡聚核苷酸引物，所述的寡聚核苷酸引物的序列如下：
FP 5’-GCTAGGTGCCGAGGGTGTT-3’
RP 5’-ACTGCAGGCTCCTTCAGAAC-3。
7.根据权利要求6所述的试剂盒，其特征在于该试剂盒还包括荧光标记探针。
8.根据权利要求6所述的试剂盒，其特征在于荧光标记探针为TaqMan MGB探针。
9.根据权利要求6所述的试剂盒，其特征在于荧光标记探针的核苷酸顺序为5’-FAM-CAAAGGTTAGGGAGGCAC-MGB-3’。
10.寡聚核苷酸引物，其特征在于：所述的寡聚核苷酸引物的序列如下：
FP 5’-GCTAGGTGCCGAGGGTGTT-3’
RP 5’-ACTGCAGGCTCCTTCAGAAC-3’。
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| CN103031368A (zh) * | 2011-09-30 | 2013-04-10 | 广州益善生物技术有限公司 | 一种fgfr1基因突变检测特异性引物和液相芯片 |
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| CN103031368A (zh) * | 2011-09-30 | 2013-04-10 | 广州益善生物技术有限公司 | 一种fgfr1基因突变检测特异性引物和液相芯片 |
| CN102352353A (zh) * | 2011-11-09 | 2012-02-15 | 湖州数问生物技术有限公司 | 一种用于pcr扩增fgfr1基因片段或者检测fgfr1基因的试剂盒和引物 |
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| CN113667758A (zh) * | 2021-10-25 | 2021-11-19 | 求臻医学科技（北京）有限公司 | 用于诊断局部晚期或转移性尿路上皮癌的组合物和试剂盒及检测方法 |
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