CN111850125A - 一种检测her2基因扩增的试剂盒及方法 - Google Patents
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Abstract
本发明的目的是提供一种基于数字微滴PCR技术检测HER2基因扩增的试剂盒及方法,以解决现有技术中HER2基因拷贝数变异判定方法准确度低的技术问题。
Description
技术领域
本发明涉及生物技术领域,基于数字微滴PCR方法检测HER2基因扩增,提供一种试剂盒及其方法。
背景技术
乳腺癌是严重威胁女性健康的恶行肿瘤,且发病率逐年上升。25%-30%的乳腺癌中有 HER2基因的扩增和蛋白的阳性表达,研究证实这部分患者OS和DFS较短,以HER2为 靶点的单克隆抗体药物赫赛汀(注射用曲妥珠单抗)现已广泛应用于临床,对乳腺癌的治 疗具有重要意义。
HER2又称为erbB2,是表皮生长因子(EGFR)家族成员之一,定位于染色体 17q12-21.32,编码185KDa的细胞受体,在肿瘤增殖、转移侵袭等方面发挥重要作用, HER2阳性的肿瘤患者对常规化疗及他莫昔芬内分泌治疗反应差,预后较差。
正确检测和评定乳腺癌的HER2蛋白表达和基因扩增状态对乳腺癌的临床治疗和预后 判断至关重要,HER2检测结果对患者临床决策起到指导作用。现行临床检测HER2推荐免疫组织化学法检测HER2蛋白表达水平,应用原位杂交法检测HER2基因扩增水平。原 位杂交法包括亮视野原位杂交法、银增强原位杂交法、荧光原位杂交法和组织原位荧光杂 交法。
数字微滴PCR(ddPCR)作为一种能对核酸进行绝对定量的方法,在肿瘤检测中广泛应用,由于其绝对定量的优势,在拷贝数变异即基因扩增检测方面具有良好的前景。
CEP17位于17p11.1-q11.1,与HER2基因定位于17号染色体临近位置,通常作为内参基因应用于HER2扩增检测中,但近年来有研究报道指出由于17号染色体着丝粒局灶性扩增导致参考基因CEP17异常扩增,HER2/CEP17作为评估HER2扩增的标准会导致一 定程度的假阴性,使一些患者失去接受进一步治疗的机会。因此,需要更精确的方法对现 有技术的检索方法进行矫正,以提高检测靶向率。
发明内容
本发明的目的是提供一种基于数字微滴PCR技术检测HER2基因扩增的试剂盒及方法, 以解决现有技术中HER2基因拷贝数变异判定方法准确度低的技术问题。
为解决上述技术问题,本发明的提供一种检测HER2基因扩增的试剂盒,其包括:
检测HER2基因扩增的第一对引物对及检测探针1,其正向引物核苷酸序列如SeqID No.1所示,即:5’-GCCCCAGCGGTGTGAA-3’,其反向引物核苷酸序列如Seq ID No.2所示,即5’-CGCCCTCCTCATCTGGAAA-3’,检测探针1序列如Seq ID No.3所示,即5’-CCTGACCTCTCCTACATGCCCATCTGG-3’,所述检测探针1为VIC荧光基团标记;
检测HER2基因扩增的第二引物对及检测探针2,其正向引物核苷酸序列如Seq IDNo.4所示,即:5’-GGCATCTGCCTGACATCCA-3’,其反向引物核苷酸序列如Seq ID No.5 所示,即5’-GCGTCCGCGGTTTTCC-3’,检测探针2序列如Seq ID No.6所示,即5’-CCCTATGGCTGCCTCTTAGACCATGTCC-3’,所述检测探针2为VIC荧光基团标记;
检测EIF2C1基因扩增的引物对及检测探针3,其正向引物核苷酸序列如Seq IDNo.7 所示,即:5’-CACGGCAAGAGATCATTGAAGAC-3’,其反向引物核苷酸序列如Seq ID No.8所示,即5’-CGGGTGGACTTGTAGAATTGG-3’,检测探针3序列如Seq ID No.9所示,即5’ -TGTCCTACATGGTGCGTGAGCTCCTC-3’,所述检测探针3为VIC荧光基团标记;
检测TFF3基因扩增的引物对及检测探针4、5,其正向引物核苷酸序列如Seq IDNo.10 所示,即:5’-TTTGTCTGGGACAGCTGCAA-3’,其反向引物核苷酸序列如Seq ID No.11所示, 即5’-GGGATCCTGGAGTCAAAGCA-3’,检测探针4和探针5序列如Seq ID No.12所示,即5’-CCCCATGTCACCCCCAAGGAGTG-3’,所述检测探针4为FAM荧光基团标记,所述检测探针5 为VIC荧光基团标记;
检测CEP17基因扩增的引物对及检测探针6、7,其正向引物核苷酸序列如Seq IDNo.13 所示,即:5’-AGTTGTGCAGCAGTTAAATTTGCT-3’,其反向引物核苷酸序列如Seq IDNo.14 所示,即5’-ACAATGCTCAACACAAAAATCAAAC-3’,检测探针6和探针7序列如Seq IDNo.15 所示,即5’-CTTAGTTCTGAAAGGTTTC-3’,所述检测探针6为FAM荧光基团标记,所述检测 探针7为VIC荧光基团标记。
本发明优选的技术方案中,所述试剂盒中优选还包括PCR预混液,dNTP。
本发明对HER2基因扩增的非诊断目的检测方法如下:
(1)提取待测样本中的DNA,
(2)以步骤(1)提取到的DNA作为模板,以本发明的基因特异性引物对以及检测探针 进行扩增,
(3)扩增结束后,检测荧光信号并使用软件对扩增产物进行拷贝数统计,HER2基因的拷贝数为HER2基因的两组引物对扩增后拷贝数的平均值并简写标记为H、CEP17基因 的拷贝数简写标记为C、EIF2C1基因的拷贝数简写标记为EI、TFF3基因的拷贝数简写标 记为TF,分别计算H/C、TF/EI、C/TF、H/TF的比值;并使用如下公式计算:
HER2copy=(HER2-1copy+HER2-2copy)/2,
内参copy=[1.04*(EIF2C1copy)+0.99*(TFF3copy)+0.97*(CEP17copy)]/3,
HER2copy/内参copy>1.85HER2基因扩增,
1.7<HER2copy/内参copy<1.85需结合其他实验判断如FISH、CISH、IHC,
HER2copy/内参copy<1.7HER2基因组拷贝数正常。
本发明优选的技术方案中,步骤(2)同时进行阴性和阳性质控品的检测。
本发明优选的技术方案中,所述样本选自血浆DNA、尿液DNA、汗液DNA、唾液DNA、精液DNA、胸水DNA、腹水DNA、粪便DNA、化石DNA、石蜡包埋DNA。
本发明优选的技术方案中,检测荧光的种类包括FAM、HEX、VIC。
本发明选取TFF3(21q22.3)、EIF2C1(1p34.3)和CEP17三个基因做为共同内参, 来增强HER2基因拷贝数变异检测结果判定的准确性。TFF3基因位于第21号染色体上; CEP17基因是着丝粒区段内的基因,作为内参基因可以解决第17号染色体出现非整倍体 和单纯的HER2基因扩增,尤其是低水平扩增的问题;EIF2C1基因位于第1号染色体上, 属于单拷贝并且保守性很强不易产生变异的基因,因此检测EIF2C1基因的拷贝数可以消除 第17号染色体发生多体现象对于HER2拷贝数变异的影响。
本发明采用数字微滴PCR检测HER2扩增,选用EIF2C1、TFF3、CEP17作为共同 内参,对EIF2C1、TFF3、CEP17基因拷贝数加权平均值进行校准,达到消除单一内参基 因拷贝数异常的影响,有助于提高HER2基因扩增的检测特异性和准确性。
本发明使用组织或循环肿瘤DNA为样本进行HER2基因扩增检测,扩增过程闭管不易 污染产生非特异扩增,检测结果与金标准的一致率达95%以上。它具有准确性好、精密度 高、操作流程简单、检测结果客观等优点。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面对本发明的具体实施方 式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发 明能够以很多不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内 涵的情况下做类似改进,因此本发明不受下面公开的具体实施例的限制。
除非另有定义,本文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。本文中在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,不是旨在于限制本发明。本文所使用的术语“及/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。
试剂与仪器
QIAgen_FFPE_DNA_kit,magmax cfDNA提取试剂盒(Thermo Fisher Scientific,MA),Covaris M220声波仪(Thermo Fisher Scientific,MA),Qubit 3.0荧光计(ThermoFisher Scientific,MA),RainDrop Digital PCR System,RainDrop微滴生成器
实施例1检测HER2基因扩增的试剂盒
1.设计并获得HER2、EIF2C1、TFF3、CEP17基因,根据现有基因数据库公布的上 述基因序列。
2.多重数字微滴PCR引物探针设计
使用primer5设计引物和探针组用来定量HER2、EIF2C1、TFF3、CEP17的DNA拷 贝数,在与常见单核苷酸多态性或拷贝数变异不相同的保守区选择目标序列。使用oligo软件对潜在的二级结构、二聚体、发卡结构等进行筛选。为了最大限度地减少多重PCR反应 中不必要的引物间相互作用,进行了碱基复合优化。
设计的引物序列如下:
3.根据上述设计的引物及探针,进行合成。
实施例2使用本发明的检测HER2基因扩增异常的试剂盒进行检测样本的HER2基因扩增异常的情况
1、取样方案的制定和游离DNA提取
样品采集和制备所有实验均按照赫尔辛基宣言和国家指导方针进行。共采集乳腺癌患 者60份血浆样本和60X10-15片病理白片(每人)。病理片使用QIAgen_FFPE_DNA_kit从5片病理片分离肿瘤DNA,30μl去离子水洗脱cfDNA,并在-80℃下保存。血浆样本使 用magmax cfDNA提取试剂盒(Thermo Fisher Scientific,MA)从2ml血浆中分离出游 离DNA(cfDNA),30μl去离子水洗脱cfDNA,并在-80℃下保存。使用Covaris M220声 波仪(ThermoFisher Scientific,MA)将肿瘤组织提取获得的DNA剪切成约200bp的片 段,放入100μlAFA Fiber Snap-Cap管中,运行以下程序:峰值入射功率50W,每次爆 发200个周期,处理时间160s,温度20℃。使用Qubit 3.0荧光计(Thermo Fisher Scientific, MA)DNA样品进行定量。
2.数字微滴PCR反应及其体系
本发明所涉及数字微滴PCR实验均在the RainDrop Digital PCR System上进行。
使用实施例1的试剂盒的引物,配置反应体系后使用RainDrop微滴生成器进行乳液生 产,提供小的反应室,反应体系如下:
PCR反应体系
PCR反应条件如下:
3.使用RainDrop数据分析软件对聚类图中的数据进行分析,包括液滴的数量、完整 性和荧光信号。质量控制标准为每50μL样品形成约400-500万个液滴,包括至少98%的完整液滴。不符合这些标准的样本数据被认为是无效的,排除在分析之外。
4.分析方法及cut-off值确定
PCR扩增后,读取并分析每滴液滴FAM和VIC荧光强度,以确定靶DNA分子的 拷贝数。并使用如下公式计算:
HER2copy=(HER2-1copy+HER2-2copy)/2
内参copy=[1.04*(EIF2C1copy)+0.99*(TFF3copy)+0.97*(CEP17copy)]/3
HER2copy/内参copy>1.85HER2基因扩增
1.7<HER2copy/内参copy<1.85需结合其他实验判断如FISH、CISH、IHC
HER2copy/内参copy<1.7HER2基因组拷贝数正常
5.检测数值
结果分析
6.结果分析
本发明所涉及HER2基因扩增数字PCR检测法能够作为FISH方法的替代,所建立EIF2C1、TFF3、CEP17三个基因拷贝数加权平均值作为内参有效可靠,与FISH方法结 果一致性高。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员 应该了解,本发明不受上述实例的限制,上述实例和说明书中描述的只是说明本发明的原 理,在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进,这些变化和改进 都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界 定。
<110> 苏州索真生物技术有限公司
<120> 一种检测HER2基因扩增的试剂盒及方法
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Claims (6)
1.一种检测HER2基因扩增的试剂盒,其特征在于,其包括:
检测HER2基因扩增的第一对引物对及检测探针1,其正向引物核苷酸序列如Seq IDNo.1所示,即:5’-GCCCCAGCGGTGTGAA-3’,其反向引物核苷酸序列如Seq ID No.2所示,即5’-CGCCCTCCTCATCTGGAAA-3’,检测探针1序列如Seq ID No.3所示,即5’-CCTGACCTCTCCTACATGCCCATCTGG-3’,所述检测探针1为VIC荧光基团标记;
检测HER2基因扩增的第二引物对及检测探针2,其正向引物核苷酸序列如Seq ID No.4所示,即:5’-GGCATCTGCCTGACATCCA-3’,其反向引物核苷酸序列如Seq ID No.5所示,即5’-GCGTCCGCGGTTTTCC-3’,检测探针2序列如Seq ID No.6所示,即5’-CCCTATGGCTGCCTCTTAGACCATGTCC-3’,所述检测探针2为VIC荧光基团标记;
检测EIF2C1基因扩增的引物对及检测探针3,其正向引物核苷酸序列如Seq ID No.7所示,即:5’-CACGGCAAGAGATCATTGAAGAC-3’,其反向引物核苷酸序列如Seq ID No.8所示,即5’-CGGGTGGACTTGTAGAATTGG-3’,检测探针3序列如Seq ID No.9所示,即5’-TGTCCTACATGGTGCGTGAGCTCCTC-3’,所述检测探针3为VIC荧光基团标记;
检测TFF3基因扩增的引物对及检测探针4、5,其正向引物核苷酸序列如Seq ID No.10所示,即:5’-TTTGTCTGGGACAGCTGCAA-3’,其反向引物核苷酸序列如Seq ID No.11所示,即5’-GGGATCCTGGAGTCAAAGCA-3’,检测探针4和探针5序列如Seq ID No.12所示,即5’-CCCCATGTCACCCCCAAGGAGTG-3’,所述检测探针4为FAM荧光基团标记,所述检测探针5为VIC荧光基团标记;
检测CEP17基因扩增的引物对及检测探针6、7,其正向引物核苷酸序列如Seq ID No.13所示,即:5’-AGTTGTGCAGCAGTTAAATTTGCT-3’,其反向引物核苷酸序列如Seq ID No.14所示,即5’-ACAATGCTCAACACAAAAATCAAAC-3’,检测探针6和探针7序列如Seq ID No.15所示,即5’-CTTAGTTCTGAAAGGTTTC-3’,所述检测探针6为FAM荧光基团标记,所述检测探针7为VIC荧光基团标记。
2.根据权利要求1所述的检测HER2基因扩增的试剂盒,其特征在于,所述试剂盒还包括PCR预混液,dNTP。
3.HER2基因扩增的非诊断目的检测方法,其特征在于,包括如下步骤:
(1)提取待测样本中的DNA,
(2)以步骤(1)提取到的DNA作为模板,以权利要求1的基因特异性引物对以及检测探针进行扩增,
(3)扩增结束后,检测荧光信号并使用软件对扩增产物进行拷贝数统计,HER2基因的拷贝数为HER2基因的两组引物对扩增后拷贝数的平均值并简写标记为H、CEP17基因的拷贝数简写标记为C、EIF2C1基因的拷贝数简写标记为EI、TFF3基因的拷贝数简写标记为TF,分别计算H/C、TF/EI、C/TF、H/TF的比值;并使用如下公式计算:
HER2 copy=(HER2-1 copy+HER2-2 copy)/2,
内参copy=[1.04*(EIF2C1 copy)+0.99*(TFF3 copy)+0.97*(CEP17 copy)]/3,
HER2 copy/内参copy>1.85HER2基因扩增,
1.7<HER2 copy/内参copy<1.85需结合其他实验判断如FISH、CISH、IHC,
HER2 copy/内参copy<1.7HER2基因组拷贝数正常。
4.根据权利要求3所述的检测方法,其特征在于,步骤(2)同时进行阴性和阳性质控品的检测。
5.根据权利要求3所述的检测方法,其特征在于,所述样本选自血浆DNA、尿液DNA、汗液DNA、唾液DNA、精液DNA、胸水DNA、腹水DNA、粪便DNA、化石DNA、石蜡包埋DNA。
6.根据权利要求3所述的检测方法,其特征在于,检测荧光的种类包括FAM、HEX、VIC。
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