CN106434913A - Erbb2基因突变检测试剂盒 - Google Patents
Erbb2基因突变检测试剂盒 Download PDFInfo
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Abstract
本发明提供一种ERBB2基因突变检测试剂盒，包括：（1）用于扩增样本中ERBB2基因Exon20号外显子的特异性引物，引物序列分别是SEQ ID Nos：1‑2；（2）用于测序PCR的测序引物，用于对ERBB2基因c.2324_2325ins12突变位点进行测序分析，引物序列是SEQ ID No.3；（3）1个装有ERBB2野生型质粒和突变型质粒按1:1质量比例混合的阳性质控品管和1个装有PCR反应预混液的试管；其中突变型质粒为ERBB2基因上的c.2324_2325ins12突变位点。本试剂盒能在样本浓度较低时检测到目的基因，结果无非特异性扩增。
Description
技术领域
本发明涉及一种试剂盒，具体涉及一种肿瘤相关基因ERBB2基因突变检测试剂盒。
背景技术
ERBB2基因最初叫NEU基因，是由Yang-Feng等于1985年从实验小鼠的恶性神经胶质细胞株中分离得到，并指出其编码一种叫p185的肿瘤相关蛋白，与表皮生长因子受体（EGFR）密切相关。同年Coussens等指出他们在细胞表面发现了一个新的酪氨酸激酶基因，并且其大部分的序列与人类上皮生长因子受体的序列相似，又因为在细胞表面接近人EGFR，所以将其命名为HER2，而Semba等鉴定了ERBB相关基因，为了别于ERBB基因，叫ERBB2基因。两年后DiFiore等指出NEU、HER2及ERBB2是同一基因，1987年Slamon等则在一项189名人乳腺癌的研究中，首次报道了ERBB2基因的扩增，并指出ERBB2基因的过表达可导致乳腺癌易复发和临床预后较差，而在正常组织中表达水平非常低。
ERBB2基因位于人17q21.1上，由922个腺嘌呤、1382个胞嘧啶、1346个鸟嘌呤和880个胸腺嘧啶组成，编码细胞膜磷酸糖蛋白，其相对分子质量(Mr)为185000，简称p185蛋白，具有跨膜酪氨酸激酶活性的生长因子受体。由胞外结构域（ECD）、跨膜结构域和细胞内结构域（ICD）三部分组成，胞外结合域主要由2个配体结合域（RLD）与2个富含半胱氨酸区构成。
迄今为止，尚未发现与ERBB2高亲和力的特异性配体，但ERBB2可与EGF受体家族的其他成员及配体形成复合物，参与细胞增殖信号的转导。ERBB2单体基本无活性，必须形成二聚体才能产生活化信号。当细胞用EGF处理后，ERBB2和EGF受体在功能上相关，起反式激活作用。EGF与EGF受体ERBB2异质二聚体结合比与EGF受体均质二聚体结合具有更高的亲和力，而且，异质二聚体比相应的均质二聚体对正常和肿瘤细胞都有更大的刺激生长活性。ERBB2是EGF受体家庭中表达更为广泛的受体，其过度表达的肿瘤细胞对化疗不敏感，它参与了抑制细胞凋亡，促进肿瘤细胞存活，上调血管内皮生长因子(VEGF)/血管通透性因子(VPF)，促进肿瘤新生血管生成，增加肿瘤细胞的侵袭力，破坏机体组织抗侵袭屏障等。因此，ERBB2是肿瘤免疫治疗研究的一个重要靶分子。
ERBB2基因突变的检测主要有Sanger测序法、ARMS、FISH等。其中FISH技术主要适用于拷贝数变异的检测，同时由于操作过程繁琐，该方法以逐渐显现其局限性；ARMS技术是目前国内应用最为广泛的技术，但只能检测已知的位点，且要将样本分成多个管进行实验才能实现分型，对样本量要求高；而Sanger测序法是DNA序列分析的经典方法，最直接的、可检测已知和未知突变的一种方法。由于该方法可直接读取DNA的序列，因此被认为是基因分型的金标准，其主要优点是测序长度较长，可发现新的变异位点，包括一些新的少见的突变形式及突变的确切类型，如点突变、片段缺失。所以探索一种可用于Sanger测序法检测ERBB2基因的技术具有重要的临床意义。
发明内容
本发明的目的是为了解决上述问题，提供一种ERBB2基因突变检测试剂盒，可直接利用含人体某组织部位的石蜡包埋样本通过Sanger测序法检测出样本中ERBB2基因主要突变位点c.2324_2325ins12。
本发明的ERBB2基因突变检测试剂盒，具体包括：
（1）用于扩增样本中ERBB2基因Exon20号外显子的特异性引物，具体序列如下:
| 名称 | 序列号 | Reverse 5’-3’ |
| ERBB2Ex20-F | SEQ ID No.1 | AATGCTTGCTCTGATAGGAAAA |
| ERBB2Ex20-R | SEQ ID No.2 | TTGTGAATACTGGGAACTATGAAAA |
（2）用于测序PCR的测序引物，用于对ERBB2基因c.2324_2325ins12突变位点进行测序分析，具体序列如下：
| 名称 | 序列号 | Reverse 5’-3’ |
| SEQ-ER20 | SEQ ID No.3 | AATGCTTGCTCTGATAGGAAAA |
（3）1个装有ERBB2野生型质粒和突变型质粒按1:1质量比例混合的阳性质控品管和1个装有PCR反应预混液的试管；其中突变型质粒为ERBB2基因上的c.2324_2325ins12突变位点；PCR反应预混液由以下组份组成：
| 组份 | 体积（μl） |
| 10PCR buffer | 2.5 |
| 25mM MgCl2 | 1.5 |
| 25mM dNTPs | 0.4 |
| H2O | 15.35 |
其中10× PCR buffer中含有500 mM KCl，100 mM Tris-HCl (pH 8.3)，100 mM (NH4)2SO4，和15 mM MgCl2；25mM dNTPs中含有25mM dATPs、25mM dTTPs、25mM dCTPs和25mMdGTPs。
本试剂盒主是根据ERBB2基因的Exon20号外显子的保守序列设计ERBB2基因c.2324_2325ins12突变位点的特异性引物，并纯化回收扩增产物。本发明的各种引物长度在19-25个碱基之间，无特殊修饰。反应液预混液采用独特的配方及比例。能在样本浓度较低时检测到目的基因，结果无非特异性扩增。具体操作流程包括：
（1）引物设计：利用引物设计软件，如Primer Premier 5，从ERBB2基因DNA序列中挑选特定的序列，然后根据碱基互补特点，设计ERBB2基因Exon20号外显子的特异性引物，用于扩增样本中DNA。测序引物的设计与用于扩增样本中DNA的特异性引物的设计类似，不过测序引物只需要一段即可，引物序列分别是SEQ ID NOs：1-3。长度在19-25个碱基左右。
（2）PCR扩增：利用试剂盒中含特定序列的引物PCR扩增临床样本中的DNA。
（3）琼脂糖凝胶电泳及胶回收：将扩增得到的目的基因ERBB2的Exon20号外显子片段回收用于测序。
附图说明
以下是附图的说明，以便于理解上述发明的目的和具体特征。
图1为 ERBB2基因的Exon20号外显子的PCR产物的琼脂糖凝胶电泳结果。
图1中各标号的具体表示如下：
1：1号样本；2：2号样本；3：3号样本；4：4号样本；5：5号样本；6：6号样本；7：7号样本；8：8号样本；M：DNA maker，从上到下大小依次为2000、1000、750、500、250和100。
图2-1为ERBB2基因的Exon20外显子的测序结果截图，测序结果为野生型。
图2-2为ERBB2基因的Exon20外显子的测序结果截图，测序结果为突变体。
具体实施方式
1、引物设计
根据ERBB2基因在肺癌等疾病中的突变情况及个体化治疗后产生的耐药机制，利用引物设计软件，如Primer Premier 5，从ERBB2基因DNA序列中挑选特定的序列，然后根据碱基互补特点，设计ERBB2基因Exon20号外显子的特异性引物，用于扩增样本中DNA。引物序列分别是SEQ ID Nos：1-2。
测序引物的设计与用于扩增样本中DNA的特异性引物的设计类似，不过测序引物只需要一段即可，引物序列分别是SEQ ID Nos：3。
2、PCR扩增
2.1、质控品准备
阳性质控品为ERBB2野生型质粒和突变型质粒按质量比1:1的比例混合，阴性质控品为无菌水。使用前室温融化，旋涡振荡10秒，瞬时离心10秒。
2.2、试剂配制
提前将试剂取出，室温融化，旋涡振荡10秒，瞬时离心10秒。
确定反应数N，N=待检样本数（n）+质控品数+1。计算加到反应混合物中的各个试剂的量，计算如下：
| 组分 | PCR mix 3（PCR反应预混液） | Taq酶 |
| 体积（μl） | 19.75×N | 0.25×N |
取一个灭菌离心管配制上述反应体系，试剂全部加入后旋涡振荡10秒，瞬时离心。然后将上述混合液按20μl/管分装至PCR反应管中。
2.3、加样
将ERBB2阳性质控品、阴性质控品和样本DNA分别取2.5 μl加入到PCR反应管中，其中样本要稀释至20ng/μl；然后再将相应的引物加入到对应的PCR反应管中，每个样本需要引物各加1.25μl（引物用之前先稀释1/10至10µM），同时作好标记，盖紧管盖后，瞬时离心15秒，将管壁上的液体全部甩至管底，消除气泡，可重复离心至气泡完全消除。然后立即进行PCR扩增反应。
2.4、PCR扩增
配置完成后，将PCR管放入PCR仪进行反应，PCR反应程序如下：
3、PCR产物的琼脂糖凝胶电泳及回收
由于PCR产物长度较短，配制2%（w/w）的琼脂糖凝胶；电泳条件为120V，20min。电泳完成后，取出凝胶，使用Bio-Rad凝胶成像系统拍照，并记录扩增条带情况。如果PCR产物电泳结果良好，即可回收目的片段用于测序。回收得到的产物即可用于测序。
序列表
<110> 中山大学 广东凯普生物科技股份有限公司
<120> ERBB2基因突变检测试剂盒
<160> 3
<210> 1
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> ERBB2Ex20-F
<400> 1
aatgcttgct ctgataggaa aa 22
<210> 2
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> ERBB2Ex20-R
<400> 2
ttgtgaatac tgggaactat gaaaa 25
<210> 3
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> SEQ-ER20
<400> 3
aatgcttgct ctgataggaa aa 22
Claims (3)
1.用于ERBB2基因突变检测的Exon20号外显子的引物，引物序列分别是SEQ ID Nos：1-3。
2.ERBB2基因突变检测试剂盒，包括：
（1）用于扩增样本中ERBB2基因Exon20号外显子的特异性引物，引物序列分别是SEQ IDNos：1-2；
（2）用于测序PCR的测序引物，用于对ERBB2基因c.2324_2325ins12突变位点进行测序分析，引物序列是SEQ ID No.3；
（3）1个装有ERBB2野生型质粒和突变型质粒按1:1质量比例混合的阳性质控品管和1个装有PCR反应预混液的试管；其中突变型质粒为ERBB2基因上的c.2324_2325ins12突变位点。
3.权利要求2所述试剂盒，其特征在于，PCR反应预混液由以下组份组成：
其中10× PCR buffer中含有500 mM KCl，100 mM Tris-HCl (pH 8.3)，100 mM (NH4)2SO4，和15 mM MgCl2；25mM dNTPs中含有25mM dATPs、25mM dTTPs、25mM dCTPs和25mMdGTPs。
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| CN112458169A (zh) * | 2020-11-25 | 2021-03-09 | 北京科途医学科技有限公司 | 用于her2基因突变检测的核酸试剂、试剂盒及检测系统 |
| WO2021059002A1 (es) * | 2019-09-25 | 2021-04-01 | Ficmac | Un método para la determinación de la inserción de 12 pares de bases en el exón 20 del gen erbb2 en muestras de tejido tumoral y adn tumoral circulante y kit de diagnóstico asociado |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021059002A1 (es) * | 2019-09-25 | 2021-04-01 | Ficmac | Un método para la determinación de la inserción de 12 pares de bases en el exón 20 del gen erbb2 en muestras de tejido tumoral y adn tumoral circulante y kit de diagnóstico asociado |
| CN111394442A (zh) * | 2020-04-30 | 2020-07-10 | 北京和合医学诊断技术股份有限公司 | 用于同步检测erbb2基因的第8、19、20、21号外显子基因突变的方法 |
| CN112458169A (zh) * | 2020-11-25 | 2021-03-09 | 北京科途医学科技有限公司 | 用于her2基因突变检测的核酸试剂、试剂盒及检测系统 |
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