WO2015180489A1 - Nras基因突变检测试剂盒 - Google Patents

Nras基因突变检测试剂盒 Download PDF

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WO2015180489A1
WO2015180489A1 PCT/CN2015/000363 CN2015000363W WO2015180489A1 WO 2015180489 A1 WO2015180489 A1 WO 2015180489A1 CN 2015000363 W CN2015000363 W CN 2015000363W WO 2015180489 A1 WO2015180489 A1 WO 2015180489A1
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gene
nras
seq
mutation
codon
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莫敏俐
李晖
陈钊
丁凤
李隽�
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Jiaxing Accb Diagnostics Ltd
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Definitions

  • the invention relates to the detection of gene mutations.
  • the invention relates to a NRAS gene mutation detection kit which can be used to detect mutations in the NRAS gene associated with cancer.
  • Ras gene is a proto-oncogene originally cloned from Harvey, Kirsten rat sarcoma, and is called HRas and KRas [1] .
  • HRas and KRas a proto-oncogene originally cloned from Harvey, Kirsten rat sarcoma
  • NRas another similar gene was discovered, which was called NRas, and the three were the most important members of the Ras gene family [2] .
  • Ras protein has the function of molecular switch, which can regulate cell growth under normal expression. Abnormalities such as point mutations, overexpression or gene translocation can lead to abnormal cell proliferation and eventually tumor formation, and more than 30% of human tumors have Ras mutations [3,4] .
  • the most in-depth study is KRas.
  • KRas is also related to TKI resistance, so it has become a molecular marker for the diagnosis and treatment of many tumors, and has played an important role in clinical [5,6] .
  • Ras Ras family
  • NRas has many similarities in structure and function to KRas, and with the deepening of research in recent years, it has gradually become another molecular indicator for clinical evaluation and treatment after KRas.
  • the NRas gene is located on the short arm of human chromosome 1 (1p22-p32) and encodes a protein of 189 amino acids p21 [7] .
  • the NRas protein shares up to 85% homology with other Ras family proteins. These highly conserved domains include the binding domains of guanosine triphosphate (GTP) and effector molecules that play an important role in protein function and will
  • GTP guanosine triphosphate
  • the NRas protein is localized on the plasma membrane of the farnesyltransferase site of action - the CAAX motif at the C-terminus [8,9] .
  • NRas also has many features common to RAS family proteins: it is located on the inner side of the cell membrane, belongs to a low molecular weight G protein, has strong affinity for guanylic acid and has GTPase activity; and has GTP binding (Ras.GTP). And GDP binding (Ras.GDP) two kinds of conformations, under certain conditions, the two can undergo mutual transformation; when Ras protein is combined with GDP, it is inactivated state, when combined with GTP, it is activated state, activates downstream signaling pathway, This plays an extremely important switching role in signal transmission [10,11] .
  • the downstream signaling pathway activated by NRAS is also the most well-defined RAS signaling pathway: PTK (protein tyrosine kinase)-Grb2 (growth factor receptor-bound protein 2)-Ras-Raf-MAPK (mitogen activated protein kinase)-ERK (extracellular signal- Regulated kinase) [12] .
  • PTK protein tyrosine kinase
  • Grb2 growth factor receptor-bound protein 2
  • Ras-Raf-MAPK mitogen activated protein kinase
  • ERP extracellular signal- Regulated kinase
  • Rho-1 ornithine exchange factor
  • Grb2-SOS The recruitment of ornithine exchange factor (SOS) binds to the SH3 region of Grb2 to form a complex, Grb2-SOS, which binds to Ras and converts Ras-GDP to Ras-GTP, thereby activating Ras.
  • Ras reactivates downstream Raf kinase
  • Raf kinase phosphorylates MAPK
  • MAPK activates ERK.
  • ERK is activated, it is transferred to the nucleus and directly activates various transcription factors of c-myc, thereby participating in various physiological processes such as cell growth, development, division and differentiation [13] .
  • NRas When NRas is mutated, it will cause abnormal activation of downstream Raf and MAPK, which plays an important role in tumor malignant transformation [14] .
  • Mutations in the Ras protein occur mainly at codons 12, 13, 59 and 61, with 12, 61 mutations occurring most frequently [15] .
  • the types of mutations are different.
  • the main mutation is that the guanine in the 12th codon is replaced by thymine, and in the same position in colon cancer, the guanine is mainly replaced by adenine [ 16] .
  • the NRas mutation occurs mainly at codon 61 and is more frequent in melanoma. Thoams et al.
  • ERK is the only known substrate for MEK, which is usually described as a single pathway downstream of Ras, it is generally believed that as long as inhibition at the MEK target is equivalent to blocking the activation of ERK caused by variant Ras, it can also be avoided. Ineffective targeting effects caused by interference from other pathways [9] .
  • NRas mutations are associated with TKI resistance in the treatment of lung cancer.
  • KRas were not detected in gefitinib-resistant PC-9 cells (PC-9/gef) compared with gefitinib-sensitive PC-9 cells (PC-9/WT) , HER2 and other EGFR-TKIs resistance genes, and the 61 codon mutation of NRas was found.
  • gefitinib or AZD6244/CI1040 alone can not promote apoptosis, the combination of the two can effectively promote apoptosis [25].
  • NRas mutations are important in the development of many tumors such as human melanoma and lung cancer.
  • the detection of mutations can accurately predict the effectiveness of the corresponding targeted drug treatment, facilitate clinical drug selection, significantly improve the treatment effect, and maximize the benefit of patients; at the same time, it can also avoid the burden of medical expenses and social medical treatment caused by irrational drug use. Waste of resources, reducing unnecessary aging losses and economic losses.
  • the invention designs a detection kit for the following detection sites:
  • the present invention relates to a NRAS gene mutation detecting kit comprising:
  • an internal reference detecting reagent comprising an internal reference gene-specific primer, an internal reference gene-specific probe, and a dNTP solution;
  • an NRAS mutation detecting reagent comprising a NRAS gene-specific primer, a NRAS gene-mutant-specific probe, an internal control gene-specific primer, an internal control gene-specific probe, and a dNTP solution;
  • PC NRAS positive control product
  • the internal reference gene-specific primers in the internal reference detection reagent in the above kit are SEQ ID No: 1 and SEQ ID No: 2; the internal reference gene-specific probe in the internal reference detection reagent is SEQ ID No: 16; the NRAS gene mutation in the NRAS mutation detection reagent is NM1, that is, the NRAS gene 12 codon 34G>A; NM2, that is, the NRAS gene 12 codon 35G>A; NM3, that is, the NRAS gene 13 codon 38G>A NM4, NRAS gene 61 codon 181C>A; NM5, ie NRAS gene 61 codon 182A>T; NM6, ie NRAS gene 61 codon 182A>G; or NM7, ie NRAS gene 61 codon 183A>T.
  • the kit of the invention can be used to detect at least one mutation in NM1, NM2, NM3, NM4, NM5, NM6 and NM7.
  • the NRAS gene mutation-specific primer in the NRAS mutation detecting reagent wherein the NRAS gene mutation-specific primer in the NRAS mutation detecting reagent is as follows: the primers for the NM1 mutation are SEQ ID No: 5 and SEQ ID No: 8; The primers for the NM2 mutation are SEQ ID No: 6 and SEQ ID No: 8; the primers for the NM3 mutation are SEQ ID No: 7 and SEQ ID No: 8; the primers for the NM4 mutation are SEQ ID No: 9 and SEQ ID No.
  • NRAS gene mutation-specific probe in the reagent is selected from the group consisting of SEQ ID No: 14 or SEQ ID No: 15; the internal control gene-specific primers in the NRAS mutation detection reagent are SEQ ID No: 3 and SEQ ID No: 4;
  • the internal control gene-specific probe in the NRAS mutation detection reagent is SEQ ID No: 17; the final concentration of the dNTP solution is 400 ⁇ M; the internal reference gene sequence is SEQ ID No: 18; and the internal control gene sequence is SEQ ID No.
  • the NRAS gene sequence is SEQ ID No: 20 or SEQ ID No: 21.
  • Figure 1 shows the results of the NM1 mutation assay in which the wild-type genomic DNA content was 20 ng/ ⁇ l, the mutant genomic DNA content was 10 ng/ ⁇ l, and contained 10, 5, 1, 0.5% mutations, respectively.
  • Figure 2 shows the results of the NM2 mutation assay in which the wild-type genomic DNA content was 20 ng/ ⁇ l, the mutant genomic DNA content was 10 ng/ ⁇ l, and contained 10, 5, 1, 0.5% mutations, respectively.
  • Figure 3 shows the results of the NM3 mutation assay in which the wild-type genomic DNA content was 20 ng/ ⁇ l, the mutant genomic DNA content was 10 ng/ ⁇ l, and contained 10, 5, 1, 0.5% mutations, respectively.
  • Figure 4 shows the results of the NM4 mutation assay in which the wild-type genomic DNA content was 20 ng/ ⁇ l, the mutant genomic DNA content was 10 ng/ ⁇ l, and contained 10, 5, 1, 0.5% mutations, respectively.
  • Figure 5 shows the results of the NM5 mutation assay in which the wild-type genomic DNA content was 20 ng/ ⁇ l, the mutant genomic DNA content was 10 ng/ ⁇ l, and contained 10, 5, 1, 0.5% mutations, respectively.
  • Figure 6 shows the results of the NM6 mutation assay in which the wild-type genomic DNA content was 20 ng/ ⁇ l, the mutant genomic DNA content was 10 ng/ ⁇ l, and contained 10, 5, 1, 0.5% mutations, respectively.
  • Figure 7 shows the results of the NM7 mutation assay in which the wild-type genomic DNA content was 20 ng/ ⁇ l, the mutant genomic DNA content was 10 ng/ ⁇ l, and contained 10, 5, 1, 0.5% mutations, respectively.
  • Real-time fluorescent PCR technology is used. Gene mutations were detected by the ARMS (amplification refractory mutation system) method. That is, by using the 3' end of the primer to recognize the mutation, the TaqMan probe is used to detect the gene mutation by hydrolyzing luminescence.
  • ARMS amplification refractory mutation system
  • the kit contains internal reference gene detection and internal control gene detection.
  • the Internal Reference is a housekeeping gene that is different from the gene to be detected, NRAS.
  • FAM channel By detecting the amplification of the internal reference gene (FAM channel), it is possible to analyze whether the DNA to be detected can be normally amplified, thereby eliminating DNA purity, poor concentration, or containing a PCR inhibitor, which causes the PCR detection failure.
  • the kit has an internal control (IC) detection system in the detection system of each mutation type of NRAS gene. Both systems react simultaneously in the same PCR tube.
  • the internal control gene is also a housekeeping gene that is different from the gene to be detected, NRAS.
  • the probe that recognizes the NRAS gene mutation template is modified to a FAM fluorescent group, and the probe that recognizes the internal control gene template is modified to a HEX fluorescent group.
  • HEX channel By detecting the internal control gene amplification (HEX channel), it can be analyzed whether the DNA to be detected can be normally amplified, thereby eliminating the cause of PCR detection failure caused by missing reagents or samples, and samples containing PCR inhibitors.
  • the internal reference gene, internal control gene fragment in the NRAS positive control (PC) can be obtained, for example, from the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/nuccore). For example, by accessing the number NG_007992.1, an amplified fragment of the reference gene (IR) can be obtained:
  • the box shows the 61 codon.
  • the four-grid method is a means well known to those skilled in the art for performing Receiver Operating Characteristic (ROC) analysis.
  • ROC Receiver Operating Characteristic
  • Eberhard DA, Johnson BE, Amler LC, etc., mutations in the epidermal growth factor receptor and KRAS are predictive and prognostic indicators for patients with non-small cell lung cancer who are treated with chemotherapy alone and in combination with erlotinib, J Clin Oncol, 2005, 23(25): 5900-5909.

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Abstract

本发明公开了一种NRAS基因突变检测试剂盒，该试剂盒可用于检测与癌症有关的NRAS基因突变。所述试剂盒包含：（1）内参检测试剂，其包含内参基因特异性引物、内参基因特异性探针及dNTP溶液；（2）NRAS突变检测试剂，其包含NRAS基因突变型特异性引物、NRAS基因突变型特异性探针、内控基因特异性引物、内控基因特异性探针及dNTP溶液；（3）Taq DNA聚合酶；和（4）NRAS阳性质控品。

Description

NRAS基因突变检测试剂盒 技术领域
本发明涉及基因突变检测。具体地说，本发明涉及NRAS基因突变检测试剂盒，该试剂盒可用于检测与癌症有关的NRAS基因突变。
背景技术
Ras基因是一种原癌基因，最初从Harvey，Kirsten大鼠肉瘤病毒(rat sarcoma)中克隆所得，被称为HRas和KRas[1]。后来将人神经母细胞瘤DNA感染NIH3T3细胞时发现另一种相似的基因，被称为NRas，三者是Ras基因家族最主要的成员[2]。功能上Ras蛋白具有分子开关的作用，正常表达下，能够调控细胞生长。发生点突变，过表达或者基因易位等异常情况会导致细胞异常增殖，并最终引发肿瘤形成，超过30％的人类肿瘤存在Ras的突变[3，4]。现在研究最为深入的是KRas，其过表达和突变普遍发生在甲状腺癌，乳腺癌等很多癌症中，在北美肺腺癌患者中，其突变率更是高达25％。并且KRas还与TKI耐药有关，所以它成为很多肿瘤诊断和治疗的分子标志，在临床上发挥了重要意义[5，6]。作为Ras家族的另一成员，NRas在结构和功能上都与KRas具有很多相似性，并随着近年研究的加深，逐渐成为继KRas之后另一个为临床病情评估和治疗提供重要依据的分子指标。
NRas基因位于人类1号染色体短臂上(1p22-p32)，编码具有189个氨基酸p21蛋白[7]。NRas蛋白与Ras家族其他蛋白具有高达85％同源性，这些高度保守的结构域包括对蛋白功能发挥起着极其重要作用的鸟苷三磷酸(guanosine triphosphate，GTP)和效应分子的结合域以及将NRas蛋白定位于质膜上的法尼基转移酶作用位点-C端的CAAX基序等[8，9]。所以功能上NRas也具有很多RAS家族蛋白共同的特征：位于细胞膜内侧，属于一种低分子量G蛋白，对鸟苷酸具有很强的亲和性并具有GTPase活力；具有GTP结合(Ras.GTP)及GDP结合(Ras.GDP)两种构象，在一定条件下二者可以发生互变；当Ras蛋白与 GDP结合时为失活状态，与GTP结合时，为活化状态，激活下游信号通路，由此在信号传导中起着极为重要的开关作用[10，11]。NRAS激活的下游信号通路也是现在研究最为明确的RAS信号通路：PTK(protein tyrosine kinase)-Grb2(growth factor receptor-bound protein2)-Ras-Raf-MAPK(mitogen activated protein kinase)-ERK(extracellular signal-regulated kinase)通路[12]。当外源刺激如生长因子(EGF)等与胞膜受体(EGFR)结合，使受体上相应的酪氨酸激酶磷酸化，磷酸化的酪氨酸残基与Grb2的SH2区结合后，招募鸟氨酸交换因子(SOS)与Grb2的SH3区结合形成复合物Grb2-SOS，该复合物与Ras结合并使Ras-GDP转变为Ras-GTP，从而激活Ras。激活后的Ras再激活下游Raf激酶，Raf激酶磷酸化MAPK，MAPK激活ERK。ERK被激活后，转至细胞核内，直接激活c-myc各种转录因子，从而参与细胞生长，发育，分裂及分化等多种生理过程[13]。当NRas发生突变时，会导致下游Raf以及MAPK等的异常激活从而在肿瘤恶变中起重要作用[14]。
Ras蛋白的突变主要发生在第12，13，59和61位密码子上，其中12，61位突变频率最高[15]。在不同癌症中，突变类型不一样，非小细胞肺癌中，主要的突变为第12位密码子中的鸟嘌呤被胸腺嘧啶取代，而在结肠癌中同一位置主要是鸟嘌呤被腺嘌呤取代[16]。NRas突变主要发生在第61位密码子，并且在黑色素瘤中频率较高。Thoams等人利用突变(mutation)和黑色素瘤(melanoma)为关键词在pubmed中检索分析了1966年-2006年发表的文章，发现在表面扩散和结节性黑色素瘤中NRas的突变率高达28％[17]。Vikas等人在60例原发性黑色素瘤中检测到10例(17％，10/16)NRas的突变，且这些突变全部为61位密码子的突变[18]。非常值得关注的是，近年NRas已被确认为肺癌发生的驱动基因。Kris等在2011年ASCO上发表了来自LCMC(NCI’s Lung Cancer Mutation Consortium)的一项研究，对1000例肺腺癌标本中KRas，EGFR，NRas等10种驱动基因进行检测，全部患者为IIIb期/IV期，并且有足够的组织标本。研究共入组830例患者，60％患者有驱动基因突变，其中NRas的突变率为0.2％[19，20]。
由于Ras突变对肿瘤的影响大多集中在Ras/Raf/MEK/ERK通路 上，所以现在针对Ras突变的靶向抗肿瘤药物主要集中在对该信号通路中的不同节点的靶向干预上。已开发的药物包括对Ras膜结合和脂质修饰进行干扰的法尼基转移酶抑制剂(FTis)，如替吡法尼(tipifarnib)等[21]，针对Raf活化的ATP间接竞争剂索拉菲尼(sorafenib)[22，23]，针对MEK靶向干预的CI-1040和AZD6244，其中后者已经进入临床试验[24]。由于ERK是目前所知的MEK唯一的底物，通常被描述为Ras下游的单一通路，所以人们一般认为只要在MEK靶点进行抑制就等于阻断了变异Ras引起的ERK的活化，还可以避免其他通路干扰所导致的无效靶向影响[9]。
需要特别指出的是，最新的研究表明，NRas突变与肺癌治疗中的TKI耐药有关。与吉非替尼(gefitinib)敏感的PC-9细胞(PC-9/WT)相比，在产生吉非替尼耐药的PC-9细胞(PC-9/gef)中并未检测到KRas，HER2等EGFR-TKIs耐药基因，而发现了NRas的61位密码子突变。并且，在吉非替尼或AZD6244/CI1040单独用药均不能促使细胞凋亡的情况下，两者联合用药时则可以有效促进细胞凋亡[25]。这些实验结果均表明，NRas突变在肺癌治疗的TKI耐药中可能发挥着重要作用，这为肺癌检测与治疗提供了新的依据和可能。
总之，越来越多的证据显示，NRas突变在人类黑色素瘤，肺癌等很多肿瘤的发生发展中具有重要意义。对其突变的检测可准确预测对应靶向药物治疗的有效性，便于临床用药选择，显著提高治疗效果，使患者最大程度受益；同时，还可以避免不合理用药造成的病人医疗费用负担及社会医疗资源浪费，减少不必要的时效损失以及经济损失。
发明内容
本发明针对以下检测位点设计了检测试剂盒：
表1.突变检测位点
编号 基因 突变位置 突变碱基 突变氨基酸
  内参基因ACTB      
NM1 NRAS 12密码子 34G＞A G12S
NM2 NRAS 12密码子 35G＞A G12D
NM3 NRAS 13密码子 38G＞A G13D
NM4 NRAS 61密码子 181C＞A Q61K
NM5 NRAS 61密码子 182A＞T Q61L
NM6 NRAS 61密码子 182A＞G Q61R
NM7 NRAS 61密码子 183A＞T Q61H
具体地说，本发明涉及一种NRAS基因突变检测试剂盒，其包含：
(1)内参检测试剂，其包含内参基因特异性引物、内参基因特异性探针及dNTP溶液；
(2)NRAS突变检测试剂，其包含NRAS基因突变型特异性引物、NRAS基因突变型特异性探针、内控基因特异性引物、内控基因特异性探针及dNTP溶液；
(3)Taq DNA聚合酶；和
(4)NRAS阳性质控品(PC)，其包含内参基因、NRAS基因突变型及内控基因片段。
更具体地说，上述试剂盒中所述内参检测试剂中内参基因特异性引物为SEQ ID No：1和SEQ ID No：2；所述内参检测试剂中内参基因特异性探针为SEQ ID No：16；所述NRAS突变检测试剂中的NRAS基因突变型为NM1，即NRAS基因12密码子34G＞A；NM2，即NRAS基因12密码子35G＞A；NM3，即NRAS基因13密码子38G＞A；NM4，即NRAS基因61密码子181C＞A；NM5，即NRAS基因61密码子182A＞T；NM6，即NRAS基因61密码子182A＞G；或NM7，即NRAS基因61密码子183A＞T。本发明的试剂盒可用于检测NM1、NM2、NM3、NM4、NM5、NM6和NM7中的至少一种突变。
所述NRAS突变检测试剂中NRAS基因突变型特异性引物其中所述NRAS突变检测试剂中NRAS基因突变型特异性引物如下：针对NM1突变的引物为SEQ ID No：5和SEQ ID No：8；针对NM2突变的引物为SEQ ID No：6和SEQ ID No：8；针对NM3突变的引物为SEQ ID No：7和SEQ ID No：8；针对NM4突变的引物为SEQ ID No：9和SEQ ID No：13；针对NM5突变的引物为SEQ ID No：10和SEQ ID No：13；针对NM6突变的引物为SEQ ID No：11和SEQ ID No：13；针对NM7突变的引物为SEQ ID No：12和SEQ ID No：13；所述NRAS突变检测 试剂中NRAS基因突变型特异性探针选自SEQ ID No：14或SEQ ID No：15；所述NRAS突变检测试剂中内控基因特异性引物为SEQ ID No：3和SEQ ID No：4；所述NRAS突变检测试剂中内控基因特异性探针为SEQ ID No：17；所述dNTP溶液的终浓度为400μM；所述内参基因序列为SEQ ID No：18；所述内控基因序列为SEQ ID No：19；所述NRAS基因序列为SEQ ID No：20或SEQ ID No：21。
附图说明
图1显示NM1突变检测的结果，其中野生型基因组DNA含量为20ng/μl，突变型基因组DNA含量为10ng/μl，并且分别含10、5、1、0.5％突变。
图2显示NM2突变检测的结果，其中野生型基因组DNA含量为20ng/μl，突变型基因组DNA含量为10ng/μl，并且分别含10、5、1、0.5％突变。
图3显示NM3突变检测的结果，其中野生型基因组DNA含量为20ng/μl，突变型基因组DNA含量为10ng/μl，并且分别含10、5、1、0.5％突变。
图4显示NM4突变检测的结果，其中野生型基因组DNA含量为20ng/μl，突变型基因组DNA含量为10ng/μl，并且分别含10、5、1、0.5％突变。
图5显示NM5突变检测的结果，其中野生型基因组DNA含量为20ng/μl，突变型基因组DNA含量为10ng/μl，并且分别含10、5、1、0.5％突变。
图6显示NM6突变检测的结果，其中野生型基因组DNA含量为20ng/μl，突变型基因组DNA含量为10ng/μl，并且分别含10、5、1、0.5％突变。
图7显示NM7突变检测的结果，其中野生型基因组DNA含量为20ng/μl，突变型基因组DNA含量为10ng/μl，并且分别含10、5、1、0.5％突变。
具体实施方式
1.实验方法
采用实时荧光PCR技术。通过ARMS(amplification refractory mutation system)方法检测基因突变。即采用引物3’端识别突变的方式，结合TaqMan探针水解发光检测基因突变。
试剂盒中含有内参基因检测，以及内控基因检测。内参基因(Internal Reference，IR)是区别于待检基因NRAS的管家基因。通过检测内参基因的扩增情况(FAM通道)，可分析待检DNA是否能被正常扩增，从而排除DNA纯度、浓度不佳，或者含有PCR抑制剂等造成PCR检测失败的原因。本试剂盒在NRAS基因各突变类型检测体系中同时设置了内控基因(Internal Control，IC)检测体系。两种体系在同一PCR管中同时进行反应。内控基因也是区别于待检基因NRAS的管家基因。将识别NRAS基因突变模板的探针修饰为FAM荧光基团，而将识别内控基因模板的探针修饰为HEX荧光基团。通过检测内控基因扩增情况(HEX通道)，可分析待检DNA是否能被正常扩增，从而排除漏加试剂或样本、样本含有PCR抑制剂等造成PCR检测失败的原因。
2.试剂盒组成(表2)：
Figure PCTCN2015000363-appb-000001
2.1 IR、NM1～7检测试剂、Taq DNA聚合酶(表3)：
Figure PCTCN2015000363-appb-000002
2.2阳性质控品PC：
人工克隆至pMD18T质粒上。
2.3引物探针序列(表4)：
Figure PCTCN2015000363-appb-000003
NRAS阳性质控品(PC)中的内参基因、内控基因片段可以从例如NCBI核苷酸数据库(http：//www.ncbi.nlm.nih.gov/nuccore)获得。例如，通过检索号NG_007992.1，可以得到内参基因(IR)的扩增片段：
CAGATGTGGATCAGCAAGCAGGAGTATGACGAGTCCGGCCCCTCCATCGTCCACCGCAAATGCTTCTAGGCGGACTATG(SEQ ID No：18)
以及内控基因(IC)的扩增片段
GATCAGCAAGCAGGAGTATGACGAGTCCGGCCCCTCCATCGTCCACCGCAAATGCTTCTAGGCGGACTATGACTTAGTTGCGTTACACC(SEQ ID No：19)。
由该数据库还可以得到含12和13密码子的NRAS的扩增片段：
GGTGGTGGTTGGAGCA
Figure PCTCN2015000363-appb-000004
GTTGGGAAAAGCGCACTGACAATCCAGCTAATCCAGAACCACTTTGTAGATGAATATGATCCCACCATAGAGGTGA(SEQ ID No：20)
其中方框显示的是12和13密码子。
由该数据库还可以得到含61密码子的NRAS的扩增片段：
ATACTGGATACAGCTGGA
Figure PCTCN2015000363-appb-000005
GAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTCTGTG(SEQ ID No：21)
其中方框显示的是61密码子。
3.实施例
3.1特异性、灵敏度：
检测20ng/μl仅含NRAS野生型的基因组DNA、10ng/μl分别含10、5、1、0.5％NRAS突变的基因组DNA(突变百分比＝突变型/野生型×100％)。实验结果见图1-7。
3.2方法对比(本发明试剂盒与测序法)：
表5.本发明试剂盒与Sanger测序法检测结果四格表
Figure PCTCN2015000363-appb-000006
其中肺癌、结直肠癌均为100例。
四格表法是本领域技术人员熟知的用于进行受试者工作特征(Receiver operating characteristic，ROC)分析的手段。
若将Sanger测序法作为检测基因突变的“金标准”，则由表5可以计算得到：NRAS试剂盒的临床灵敏度＝3/(3+0)＝100％，临床特异性＝196/(1+196)＝99.5％，总体一致性＝(3+196)/(3+196+1+0)＝99.5％。由此可见，本发明的试剂盒具有很高的临床灵敏度、临床特异性、总体一致性。
另外，从四格表数据可见，Sanger测序法阳性的样本，用定量PCR(QPCR)检测均为阳性，但用定量PCR检测为阳性的样本，可能用Sanger测序法检测为阴性，表明本发明的定量PCR试剂盒灵敏度较Sanger测序法的灵敏度高。
参考文献
[1]Ellis R.W.，DeFeo D.，Furth M.E.等，小鼠细胞包含两个不同的ras基因mRNA物类，可被翻译为p21 onc蛋白，Mol Cell Biol，1982，2(11)：1339-1345。
[2]Stacey D.W.，Kung H.F.，通过微注射Ha-ras p21蛋白转化NIH3T3细胞，Nature，1984，310(5977)：508-511。
[3]Baines A.T.，Xu D.，Der C.J.，用于癌症治疗的Ras抑制：研究在继续，Future Med Chem，2011，3(14)：1787-1808。
[4]Overmeyer J.H.，Maltese W. A.，癌细胞中活化的Ras引发的死亡通路，Front Biosci(Landmark Ed)，2011，16：1693-1713。
[5]Shigematsu H.，Gazdar A.F.，肺癌中表皮生长因子受体信号通路的体细胞突变，Int J Cancer，2006，118(2)：257-262。
[6]Eberhard D.A.，Johnson B.E.，Amler L.C.等，表皮生长因子受体和KRAS中的突变是单独实施化疗以及化疗与厄洛替尼联合施用的非小细胞肺癌患者的预测和预后指标，J Clin Oncol，2005，23(25)：5900-5909。
[7]Rabin M.，Watson M.，Barker P.E.等，通过原位杂交将染色体1上的基因图谱NRAS转化至区域p11----p13，Cytogenet Cell Genet，1984， 38(1)：70-72。
[8]Chetty R.，Govender D.，本月基因：KRAS，J Clin Pathol，2013，66(7)：548-550。
[9]昌毓穗刘，傅华群，喻本桐，邹书兵，吴起才，万力，Ras/Raf/MEK/ERK通路在食管癌药物靶向治疗中的作用[J].药学学报Acta Pharmaceutica Sinica，2013，48(5)：635-641。
[10]Shih T.Y.，Hattori S.，Clanton D.J.等，p21 ras蛋白的结构和功能，Gene Amplif Anal，1986，4：53-72。
[11]Lacal J.C.，Anderson P.S.，Aaronson S.A.，Harvey ras p21蛋白缺失突变揭示了至少两个不同的区域对GTP结合和转化活性是绝对必要的，EMBO J，1986，5(4)：679-687。
[12]Marais R.，Marshall C.J.，通过Ras和Raf控制ERK MAP激酶级联，Cancer Surv，1996，27：101-125。
[13]Andresen B.T.，Rizzo M.A.，Shome K.等，磷脂酸在调节Ras/MEK/Erk信号级联中的作用，FEBS Lett，2002，531(1)：65-68。
[14]Bauer J，Curtin J.A.，Pinkel D.等，Congenital先天黑色素痣通常锚定NRAS突变而不是BRAF突变，J Invest Dermatol，2007，127(1)：179-182。
[15]Smit V. T.，Boot A.J.，Smits A.M.等，胰腺瘤中普遍存在KRAS密码子12突变，Nucleic Acids Res，1988，16(16)：7773-7782。
[16]何太平，严卫红.Ras信号通路转导[J].国外医学临床生物化学与检验学分册，2004，25(1)：74-76。
[17]Hocker T.，Tsao H.，紫外辐射和黑素瘤：报告的序列变体的系统性综述和分析，Hum Mutat，2007，28(6)：578-588。
[18]Goel V. K.，Lazar A.J.，Warneke C.L.等，原发性皮肤黑素瘤中BRAF、NRAS和PTEN突变的研究，J Invest Dermatol，2006，126(1)：154-160。
[19]Kris MG J.B.，Kwiatkowski D.G.等，来自1000位肺腺癌患者的肿瘤样本中驱动突变(driver mutations)的鉴定，The NCI’s Lung Cancer Mutation Consortium(LCMC)J Clin Oncol，2011，29(18)：abstract 7506。
[20]王敬慧，张宗德，张树才.肺腺癌驱动基因研究相关进展[J].中 国肺癌杂志，2013，16(2)：91-96。
[21]Medeiros B.C.，Landau H.J.，Morrow M.等，法呢基转移酶抑制剂替吡法尼是MDR1基因产物P-糖蛋白的强力抑制剂，并且表现出抗人白血病细胞系的显著的细胞毒性协同作用，Leukemia，2007，21(4)：739-746。
[22]蒋成英.R as R af MekErk信号传导通路在肝细胞癌发生中的作用机制及在靶向治疗中的应用[J].中国肿瘤临床，2008，35(23)：1377-1380。
[23]Keswani R.N.，Chumsangsri A.，Mustafi R.等，索拉菲尼在巴雷特食管腺癌细胞系中抑制MAPK介导的增殖，Dis Esophagus，2008，21(6)：514-521。
[24]Martin T.D.，Samuel J.C.，Routh E.D.等，Ral GTPases在直肠癌中的激活和作用，Cancer Res，2011，71(1)：206-215。
[25]Huang M.H.，Lee J.H.，Chang Y. J.等，在具有获得性吉非替尼耐受的表皮生长因子受体突变肺癌细胞中EK抑制剂的抗逆性，Mol Oncol，2013，7(1)：112-120。

Claims (3)

  1. 一种NRAS基因突变检测试剂盒，其包含：
    (1)内参检测试剂，其包含内参基因特异性引物、内参基因特异性探针及dNTP溶液，
    其中所述内参基因特异性引物为SEQ ID No：1和SEQ ID No：2，所述内参基因特异性探针为SEQ ID No：16；
    (2)NRAS突变检测试剂，其包含NRAS基因突变型特异性引物、NRAS基因突变型特异性探针、内控基因特异性引物、内控基因特异性探针及dNTP溶液，
    其中所述NRAS基因突变型选自：
    NM1，即NRAS基因12密码子34G＞A；
    NM2，即NRAS基因12密码子35G＞A；
    NM3，即NRAS基因13密码子38G＞A；
    NM4，即NRAS基因61密码子181C＞A；
    NM5，即NRAS基因61密码子182A＞T；
    NM6，即NRAS基因61密码子182A＞G；和
    NM7，即NRAS基因61密码子183A＞T；
    针对NM1突变的所述NRAS基因突变型特异性引物为SEQ ID No：5和SEQ ID No：8；
    针对NM2突变的所述NRAS基因突变型特异性引物为SEQ ID No：6和SEQ ID No：8；
    针对NM3突变的所述NRAS基因突变型特异性引物为SEQ ID No：7和SEQ ID No：8；
    针对NM4突变的所述NRAS基因突变型特异性引物为SEQ ID No：9和SEQ ID No：13；
    针对NM5突变的所述NRAS基因突变型特异性引物为SEQ ID No：10和SEQ ID No：13；
    针对NM6突变的所述NRAS基因突变型特异性引物为SEQ ID No：11和SEQ ID No：13；
    针对NM7突变的所述NRAS基因突变型特异性引物为SEQ ID No： 12和SEQ ID No：13；
    所述NRAS基因突变型特异性探针选自SEQ ID No：14或SEQ ID No：15；
    所述内控基因特异性引物为SEQ ID No：3和SEQ ID No：4；
    所述内控基因特异性探针为SEQ ID No：17；
    (3)Taq DNA聚合酶；和
    (4)NRAS阳性质控品，其包含内参基因、NRAS基因突变型及内控基因片段，
    其中所述内参基因序列为SEQ ID No：18；
    所述内控基因片段序列为SEQ ID No：19；
    NRAS基因序列为SEQ ID No：20或SEQ ID No：21。
  2. 权利要求1所述的试剂盒，其中所述探针的5’端连接FAM基团，3’端连接BHQ 1基团。
  3. 权利要求1所述的试剂盒，其中所述dNTP溶液的终浓度为400μM。
PCT/CN2015/000363 2014-05-30 2015-05-27 Nras基因突变检测试剂盒 Ceased WO2015180489A1 (zh)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110157593A (zh) * 2018-04-13 2019-08-23 左雨萌 一种dna基因突变检测试剂盒
CN112837749A (zh) * 2021-02-01 2021-05-25 北京百奥纳芯生物科技有限公司 一种癌症筛查用基因芯片探针的优选方法
CN113897429A (zh) * 2020-07-06 2022-01-07 铭炽生物科技(上海)有限公司 人nras基因突变的数字pcr检测方法及应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104232750A (zh) * 2014-05-30 2014-12-24 嘉兴雅康博医学检验所有限公司 Nras基因突变检测试剂盒
CN105986018A (zh) * 2015-02-06 2016-10-05 上海赛安生物医药科技有限公司 Nras基因突变检测体系及其试剂盒
CN107164496A (zh) * 2017-06-06 2017-09-15 上海安甲生物科技有限公司 与甲状腺癌相关的基因多态性位点及其应用
CN107937525B (zh) * 2017-12-08 2020-10-30 益善生物技术股份有限公司 基于液相芯片法的nras突变检测试剂盒和延伸引物
CN112176062A (zh) * 2020-10-13 2021-01-05 苏州中科先进技术研究院有限公司 检测nras基因突变的核酸组合物及其试剂盒
CN115029440A (zh) * 2022-06-24 2022-09-09 湖南圣洲生物科技有限公司 一种检测人nras基因突变的引物、探针和试剂盒

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102301005A (zh) * 2008-12-17 2011-12-28 生命技术公司 用于检测等位基因变体的方法、组合物和试剂盒
CN102676648A (zh) * 2011-03-07 2012-09-19 重庆医科大学附属儿童医院 白血病诊疗的基因芯片
CN103703013A (zh) * 2011-02-14 2014-04-02 斯威夫特生物科学公司 多核苷酸引物和探针
CN104232750A (zh) * 2014-05-30 2014-12-24 嘉兴雅康博医学检验所有限公司 Nras基因突变检测试剂盒

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100286143A1 (en) * 2009-04-24 2010-11-11 Dora Dias-Santagata Methods and materials for genetic analysis of tumors
CN102453748A (zh) * 2010-10-18 2012-05-16 北京雅康博生物科技有限公司 用于荧光定量pcr定量检测的质粒标准品
CN102994619B (zh) * 2011-09-13 2014-12-10 益善生物技术股份有限公司 一种nras基因突变检测特异性引物和液相芯片
US9260714B2 (en) * 2011-12-02 2016-02-16 Roche Molecular Systems, Inc. Suppression of non-specific amplification with high-homology oligonucleotides
CN102925562B (zh) * 2012-10-17 2014-02-12 亚能生物技术（深圳）有限公司 氨基糖苷类药物性耳聋易感基因检测试剂盒及方法
CN103305625B (zh) * 2013-07-08 2014-06-25 广东省人民医院 检测非小细胞肺癌驱动基因突变谱的方法及试剂盒与应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102301005A (zh) * 2008-12-17 2011-12-28 生命技术公司 用于检测等位基因变体的方法、组合物和试剂盒
CN103703013A (zh) * 2011-02-14 2014-04-02 斯威夫特生物科学公司 多核苷酸引物和探针
CN102676648A (zh) * 2011-03-07 2012-09-19 重庆医科大学附属儿童医院 白血病诊疗的基因芯片
CN104232750A (zh) * 2014-05-30 2014-12-24 嘉兴雅康博医学检验所有限公司 Nras基因突变检测试剂盒

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3150719A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110157593A (zh) * 2018-04-13 2019-08-23 左雨萌 一种dna基因突变检测试剂盒
CN113897429A (zh) * 2020-07-06 2022-01-07 铭炽生物科技(上海)有限公司 人nras基因突变的数字pcr检测方法及应用
CN112837749A (zh) * 2021-02-01 2021-05-25 北京百奥纳芯生物科技有限公司 一种癌症筛查用基因芯片探针的优选方法
CN112837749B (zh) * 2021-02-01 2021-11-26 北京百奥纳芯生物科技有限公司 一种癌症筛查用基因芯片探针的优选方法
US11710537B2 (en) 2021-02-01 2023-07-25 Beijing Bionaxin Biotech Co., Ltd Optimal selection method of gene chip probes for cancer screening

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