WO2017000534A1 - 用于检测c-kit基因突变的引物、探针及试剂盒 - Google Patents

用于检测c-kit基因突变的引物、探针及试剂盒 Download PDF

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WO2017000534A1
WO2017000534A1 PCT/CN2016/000352 CN2016000352W WO2017000534A1 WO 2017000534 A1 WO2017000534 A1 WO 2017000534A1 CN 2016000352 W CN2016000352 W CN 2016000352W WO 2017000534 A1 WO2017000534 A1 WO 2017000534A1
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kit
seq
gene
mutation
probe
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莫敏俐
丁凤
陈钊
李晖
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嘉兴雅康博医学检验所有限公司
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Priority claimed from CN201510364304.0A external-priority patent/CN106319036A/zh
Priority claimed from CN201510364303.6A external-priority patent/CN106319035A/zh
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  • the present invention relates to the field of biotechnology, and in particular to a primer, a probe and a related kit for detecting a mutation of a C-KIT gene.
  • C-KIT is a protooncogene located on chromosome 4q11-12 with a total length of about 80 kb.
  • the C-KIT-encoded C-KIT protein is a member of the type III receptor tyrosine kinase family, a transmembrane protein with a molecular weight of approximately 145 kD, comprising an intracellular tyrosine kinase domain, a transmembrane domain, and ligand binding. Site extracellular region.
  • the ligand binds to it, it activates its own tyrosine protein kinase activity, and activates the downstream signal transduction pathway through a series of reactions, thereby regulating cell growth and proliferation.
  • Gastrointestinal stromal tumors are the most common mesenchymal tumors of the digestive tract. Studies have shown that the C-KIT gene mutation rate in GIST patients is about 90%.
  • the inventors of the present invention found a new mutation site of the C-KIT gene associated with gastrointestinal stromal tumors, namely 1672-1713 deletion mutation, in the routine detection of gastrointestinal stromal tumors (GIST), and The 1700-1733 position was replaced by a mutation of T.
  • the inventors of the present invention have developed a rapid, highly sensitive, and easy-to-use detection kit and related detection method for the mutation, which can be used for the prognosis of GIST chemotherapy.
  • the object of the present invention is to provide a primer and a probe for detecting a mutation of a C-KIT gene which is a deletion of a 42 base mutation of 1672-1713 (1672_1713del 42, K558_I571del), or a substitution of 1700-1733 to T Mutation (1700_1733>T, N567_Y578>I).
  • the primers and probes of the invention comprise the following sequences:
  • Reverse primer GACCAAAAACTCAGCCTGT (SEQ ID No: 2)
  • Another aspect of the present invention provides a detection kit for detecting a mutation of a C-KIT gene, the kit comprising the primer shown in SEQ ID No. 1 - SEQ ID No. 2 and the SEQ ID No. 3 The probe or the primer shown in SEQ ID No. 4-SEQ ID No. 5 and the probe shown in SEQ ID No. 6.
  • the kit of the present invention may further comprise an internal reference gene and an internal control gene depending on the PCR method employed.
  • Another aspect of the invention provides a method of detecting a C-KIT gene mutation comprising the steps of:
  • the detection sample includes fresh pathological tissue, paraffin-embedded tissue, whole blood and plasma;
  • the present invention employs specific primer and probe technologies to specifically detect C-KIT gene mutations. This method has high sensitivity, high specificity and fast detection speed.
  • Figure 1 is a PCR diagram of a negative sample detected using the sequence of SEQ ID No. 1-3.
  • Figure 2 is a PCR diagram for detecting a mutation-positive sample using the sequence of SEQ ID No. 1-3.
  • Figure 3 is a PCR diagram of a negative sample detected using the sequence of SEQ ID No. 4-6.
  • Figure 4 is a PCR diagram for detecting a mutation-positive sample using the sequence of SEQ ID No. 4-6.
  • Taq DNA polymerase uses deoxynucleotides (dNTPs) as a substrate, and internal reference gene (Internal Reference, IR) and C-KIT gene mutant genes are in vitro. Amplification. The detection was carried out by fluorescence PCR, and the fluorescence was released by specific probe hydrolysis, and the progress of the PCR reaction was monitored to determine the mutation of the C-KIT gene.
  • dNTPs deoxynucleotides
  • IR Internal Reference, IR
  • C-KIT gene mutant genes are in vitro. Amplification. The detection was carried out by fluorescence PCR, and the fluorescence was released by specific probe hydrolysis, and the progress of the PCR reaction was monitored to determine the mutation of the C-KIT gene.
  • the kit of the present invention is separately provided with an internal reference gene detection system.
  • the internal reference gene is a housekeeping gene that is different from the C-KIT gene to be examined. By detecting the amplification of the internal reference gene (FAM channel), it can be analyzed whether the DNA to be detected can be normally amplified, thereby eliminating DNA purity, poor concentration, or containing PCR inhibitors and the like to cause PCR detection failure.
  • the kit of the present invention simultaneously sets an internal control (IC) detection system in the C-KIT gene mutation detection system. Both systems react simultaneously in the same PCR tube.
  • the internal control gene is also a housekeeping gene that is different from the gene C-KIT to be detected.
  • the probe that recognizes the C-KIT 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 possibility of PCR detection failure caused by missing reagents or samples, and samples containing PCR inhibitors.
  • the FAM and HEX channels in the negative control (NC) should be amplified without a typical S-shaped curve.
  • the typical S-shaped curve is in the exponential phase and straight line.
  • Period and platform period) or no Ct value, FAM, HEX channel detection in positive control products (PC) should have amplification (typical S-shaped curve) and Ct value ⁇ 28; otherwise, the experiment is invalid, repeat experiment .
  • the optimized primers and probes are as follows:
  • Reverse primer GACCAAAAACTCAGCCTGT (SEQ ID No: 2)
  • Probe FAM-TCTGGGAAACTCCCATTTGTGATCA-BHQ-1 (SEQ ID No: 3, with fluorescent label)
  • Probe FAM-CTCAGCCTGTTTCTGGGAAACTCCC-BHQ-1 (SEQ ID No: 6, with fluorescent label)
  • the test sample may be fresh pathological tissue, paraffin embedded tissue, whole blood, plasma, and peritoneal effusion.
  • the following is only an example of a paraffin-embedded tissue sample.
  • the patient was not treated with tyrosine kinase inhibitors (TKIs); since the quality of the DNA sample will affect the test results, it should be determined that the paraffin-embedded tissue sample from the DNA sample contains cancerous tissue.
  • the paraffin-embedded tissue samples should not be stored for more than 12 months at room temperature, and the extracted DNA samples should be stored under -20 °C freezing conditions for a period of not more than 6 months.
  • the composition of the kit is shown in Table 1.
  • the kit does not contain nucleic acid extraction components, and DNA extraction of paraffin-embedded tissue samples is performed using the QIAamp DNA FFPE Tissue Kit (Qiagen, Cat. No. 56404) kit.
  • Detection reagent composition and C-KIT gene to be tested for mutation sites See Table 2.
  • the IR detection reagent contains only the internal reference gene detection system (FAM channel), and the C-KIT detection reagent also contains the C-KIT gene mutation detection system (FAM channel) and the internal control gene detection system (HEX channel).
  • the specific sequence of the internal reference gene and the internal control gene can be easily determined by a person skilled in the art according to experimental conditions or provided by Beijing Yakambo Biotechnology Co., Ltd.
  • test reagent and positive control PC
  • the test reagent and the positive control product PC were briefly centrifuged and placed on ice.
  • the IR detection reagent and Taq DNA polymerase were mixed at a volume ratio of 17:0.3, and the C-KIT detection reagent and Taq DNA polymerase were mixed at a volume ratio of 17:0.3.
  • the IR detection reagent and C-KIT detection reagent were dispensed into the eight tubes at 17.3/well.
  • PC blow and mix, cover the tube cover and centrifuge briefly. Note: Do not use a vortex oscilloscope when mixing; follow up immediately after mixing.
  • Fluorescence PCR instrument detection channel setting needs to select FAM, HEX channel at the same time (reference The dye is set to "None").
  • the reaction procedure is set as follows (Table 2):
  • Ct value determination First set the baseline of the Stratagene MX3000P fluorescence PCR instrument: select the fluorescence signal when the "Adaptive baseline” setting is selected, and the threshold setting principle is just above the threshold line just above the normal negative control. The highest point of the NC amplification curve (random noise line) is that the NC control curve of the negative control shows "No Ct". The Ct value of each sample detected at each point is read from the software.
  • Figures 1 and 3 are PCR diagrams of samples with negative detection results
  • Figures 2 and 4 are PCR diagrams of samples with positive detection results.
  • the fluorescent PCR reaction system of the present invention can detect a 42 base mutation of the C-KIT gene 1672-1713, or a mutation of the C-KIT gene 1700-1733 to a T, which is convenient, rapid, and accurate. The requirement for rapid detection of C-KIT gene mutations.

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Abstract

本发明公开了用于检测C-KIT基因突变的引物、探针及试剂盒。所述引物如SEQ ID No.1-SEQ ID No.2所示，或者如SEQ ID No.4-SEQ ID No.5所示，所述探针如SEQ ID No.3所示，或者如SEQ ID No.6所示。利用本发明的试剂盒进行实时荧光PCR，可以检测C-KIT基因1672-1713位缺失突变，或者检测C-KIT基因1700-1733位替换为T的突变。

Description

用于检测C-KIT基因突变的引物、探针及试剂盒 技术领域
本发明涉及生物技术领域，具体地说，涉及一种用于检测C-KIT基因突变的引物、探针及相关试剂盒。
背景技术
C-KIT是一种原癌基因，位于染色体4q11-12，全长约80kb。C-KIT编码的C-KIT蛋白属于III型受体酪氨酸激酶家族成员，它是分子量约为145KD的跨膜蛋白，包含了胞内的酪氨酸激酶区、跨膜区和配体结合位点胞外区。当配体与其结合后能激活自身酪氨酸蛋白激酶活性，通过一系列反应激活下游信号转导通路，从而调节细胞的生长与增殖。
胃肠道间质瘤(GIST)是消化道最常见的间叶源性肿瘤。研究表明，在GIST患者中C-KIT基因突变率约为90％。
目前Sanger测序法是C-KIT基因突变检测的主要方法。然而，该方法的灵敏度低，会导致漏检及假阴性的发生，而且检测时间长，无法满足临床检测的实际需求。临床上迫切需要开发一种高灵敏度、快速的C-KIT基因突变检测技术。
本发明的发明人在胃肠道间质瘤(GIST)的日常检测中发现了与胃肠道间质瘤有关的位于C-KIT基因的新突变位点，即1672-1713位缺失突变，以及1700-1733位替换为T的突变。本发明的发明人针对该突变开发了一种快速、高灵敏、操作简便的检测试剂盒及相关的检测方法，可以用于GIST化疗预后。
发明内容
本发明的目的在于提供用于检测C-KIT基因突变的引物和探针，所述突变是1672-1713位缺失42碱基突变(1672_1713del 42，K558_I571del)，或者是1700-1733位替换为T的突变(1700_1733＞T，N567_Y578＞I)。本发明的引物与探针包括如下序列：
对于1672-1713位缺失突变：
正向引物(F)：AAGTACAGTGGGACCCAA(SEQ ID No：1)
反向引物(R)：GACCAAAACTCAGCCTGT(SEQ ID No：2)
探针(PB)：TCTGGGAAACTCCCATTTGTGATCA(SEQ ID No：3)
对于1700-1733位替换为T的突变：
正向引物(F)：GAGATAAATGGAAACATTGATCAC(SEQ ID No：4)
反向引物(R)：CCCTGTTTCATACTGACCAA(SEQ ID No：5)
探针(PB)：CTCAGCCTGTTTCTGGGAAACTCCC(SEQ ID No：6)
本发明另一方面提供一种用于检测C-KIT基因突变的检测试剂盒，所述试剂盒包括SEQ ID No.1-SEQ ID No.2所示的引物和SEQ ID No.3所示的探针，或者包括SEQ ID No.4-SEQ ID No.5所示的引物和SEQ ID No.6所示的探针。
根据所采用的PCR方法，本发明的试剂盒还可以包括内参基因和内控基因。
本发明另一方面提供一种检测C-KIT基因突变的方法，其包括以下步骤：
(1)提取样本中的基因组DNA，检测样本包括新鲜病理组织、石蜡包埋组织、全血和血浆等；
(2)用本发明的试剂盒对DNA进行扩增；
(3)根据荧光PCR仪显示的Ct值判断检测结果：检测反应体系的FAM荧光强度，以FAM达到设定的阈值时所需要的循环次数Ct值作为阴性、阳性判定标准，Ct值＞38或无扩增为阴性，Ct值≤38为阳性。
本发明采用了特异性引物和探针技术，可以特异性检测C-KIT基因突变。此方法灵敏度高、特异性强、检测速度快。
附图说明
图1为用SEQ ID No.1-3序列检测阴性样本的PCR图。
图2为用SEQ ID No.1-3序列检测突变阳性样本的PCR图。
图3为用SEQ ID No.4-6序列检测阴性样本的PCR图。
图4为用SEQ ID No.4-6序列检测突变阳性样本的PCR图。
具体实施方式
下面结合具体实施例，对本发明进一步阐述。应理解，这些实施例仅用于本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法，按照本领域技术人员熟知的常规条件，或者按照制造厂商建议的条件。
一.原理
本发明的特异性引物与探针同DNA模板结合后，Taq DNA聚合酶以脱氧核苷酸(dNTP)为底物，对内参基因(Internal Reference，IR)及C-KIT基因突变型基因进行体外扩增。检测采用荧光PCR技术，通过特异性探针水解释放荧光，监测PCR反应的进行，确定C-KIT基因突变情况。
本发明的试剂盒单独设置了内参基因检测体系。内参基因是区别于待检C-KIT基因的管家基因。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检测失败的情况。
本发明的试剂盒在C-KIT基因突变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C-KIT的管家基因。将识别C-KIT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检测失败的情况。
进行本试剂盒检测结果判定时，阴性质控品(NC)中FAM、HEX通道检测均应无扩增(不呈典型的S型曲线。注：典型的S型曲线依次表现为指数期、直线期及平台期)或无Ct值，阳性质控品(PC)中FAM、HEX通道检测均应有扩增(呈典型的S型曲线)且Ct值≤28；否则认为实验无效，需重复实验。
二.实验材料和设备
1.针对突变位点设计合成特异性引物和探针
针对C-KIT基因突变位点设计特异引物和探针。通过特异性引物和探针优化，以便实现高灵敏和快速检测。
优化后的引物与探针如下：
正向引物(F)：AAGTACAGTGGGACCCAA(SEQ ID No：1)
反向引物(R)：GACCAAAACTCAGCCTGT(SEQ ID No：2)
探针(PB)：FAM-TCTGGGAAACTCCCATTTGTGATCA-BHQ-1(SEQ ID No：3，含荧光标记)
或者为：
正向引物(F)：GAGATAAATGGAAACATTGATCAC(SEQ ID No：4)
反向引物(R)：CCCTGTTTCATACTGACCAA(SEQ ID No：5)
探针(PB)：FAM-CTCAGCCTGTTTCTGGGAAACTCCC-BHQ-1(SEQ ID No：6，含荧光标记)
2.检测样本处理与DNA的提取
检测样本可以是新鲜病理组织、石蜡包埋组织、全血、血浆和腹腔积液。以下仅以石蜡包埋组织样本为例进行说明。
在样本采集之前，病人未经过酪氨酸激酶抑制剂(tyrosine kinase inhibitors，TKIs)类药物治疗；由于DNA样品质量会影响检测结果，因此应确定DNA样品来源的石蜡包埋组织样本中含有癌组织细胞，并且不少于整个样本的25％；且DNA样品OD260/OD280＝1.8±0.2，OD260/OD230≥1.7，浓度为5-10ng/μl。
石蜡包埋组织样本在室温下保存时限不超过12个月，提取后的DNA样品在-20℃冷冻条件下保存时限不超过6个月。
3.适用仪器
Stratagene Mx3000P。
4.试剂盒的组成
试剂盒组成见表1。试剂盒中不含核酸提取成份，使用QIAamp DNA FFPE Tissue Kit(Qiagen公司生产，货号：56404)试剂盒完成石蜡包埋组织样本DNA提取。检测试剂组成及C-KIT基因待检突变位点 见表2。IR检测试剂只含内参基因检测体系(FAM通道)，C-KIT检测试剂同时含有C-KIT基因突变检测体系(FAM通道)及内控基因检测体系(HEX通道)。
表1：试剂盒组成
Figure PCTCN2016000352-appb-000001
其中所述内参基因和内控基因的具体序列可以由本领域技术人员根据实验情况容易地确定或者由北京雅康博生物科技有限公司提供。
三.检测方法
1.使用QIAamp DNA FFPE Tissue Kit(Qiagen公司生产)试剂盒，按照使用说明书进行石蜡包埋组织样本DNA提取。
2.取试剂盒中的检测试剂以及阳性质控品(PC)。待检测试剂及阳性质控品PC融化后短暂离心，置于冰上。
3.按17∶0.3的体积比例混合IR检测试剂和Taq DNA聚合酶，按17∶0.3的体积比例混合C-KIT检测试剂和Taq DNA聚合酶。按17.3/孔分装IR检测试剂和C-KIT检测试剂至八连管中。向装有IR检测试剂和C-KIT检测试剂的八连管中分别加入2.7μl待检DNA样品、阴性质控品(NC，溶解DNA的缓冲液，由使用者自备)及阳性质控品PC，吹打混匀，盖紧管盖后短暂离心。注意：混匀时不要使用漩涡振荡仪；混匀后立即进行后续操作。
4.荧光PCR仪检测通道设定需同时选择FAM、HEX通道(参比 染料设置为“无”)。反应程序设置如下(表2)：
表2：
Figure PCTCN2016000352-appb-000002
四.检测结果的解释
1.Ct值确定：首先设定Stratagene MX3000P荧光PCR仪的基线：选择“适合基线(Adaptive baseline)”设定时的荧光信号，阈值(threshold)设定原则以阈值线刚好超过正常阴性质控品NC扩增曲线(无规则的噪音线)的最高点，即令阴性质控品NC扩增曲线显示“No Ct”为准。从软件中读取各样本在各位点检测的Ct值。
2.定性分析：
(1)实验质量评判：若NC中FAM、HEX通道检测均无扩增(不呈典型的S型曲线。注：典型的S型曲线依次表现为指数期、直线期及平台期)或无Ct值，PC中FAM、HEX通道检测均有扩增(呈典型的S型曲线)且Ct值≤28，则可继续分析；否则认为实验无效，需重复实验。
(2)待检DNA样品中内参基因(IR)检测情况评判：若内参基因检测(FAM通道)有扩增且19≤Ct值≤25，则可继续分析；若其Ct值较小，则认为DNA样品浓度过高；若其Ct值较大或无扩增，则认为DNA样品浓度过低、发生降解，或其中可能含有PCR抑制剂。
(3)待检DNA样品中内控基因检测情况评判：若内控基因检测(HEX通道)有扩增且Ct值≤25，则可继续分析；若内控基因检测(HEX通道)Ct值较大或无扩增，但突变位点检测(FAM通道)有扩增且Ct值≤38，则可继续分析(可能由于突变位点扩增对内控基因扩增产生抑制)；若内控基因检测(HEX通道)Ct值较大或无扩增，而突变位点检测(FAM通道)无扩增或有扩增但Ct值＞38，则无法继续分析，需重复实验(可能由于DNA样品发生降解、其中含有PCR抑制剂或未加样品)。
(4)待检DNA样品中基因突变情况评判：若样品中某突变位点检测(FAM通道)有扩增且Ct值≤38，则判定该样本突变结果为阳性；若Ct值＞38或无扩增，则判定该样本突变结果为阴性。
注意：同一DNA样品中可能同时存在多种突变。
图1和图3为检测结果为阴性的样本的PCR图，图2和图4为检测结果为阳性的样本的PCR图。
通过对比检测，证实本发明的荧光PCR方法和传统测序方法的结果是相符的，而本发明的荧光PCR方法的灵敏度和选择性高于传统测序方法。
因此，本发明荧光PCR反应体系可检测C-KIT基因1672-1713位缺失42碱基突变，或者检测C-KIT基因1700-1733位替换为T的突变，检测方便快捷，准确性高，可满足C-KIT基因突变快速检测的要求。

Claims (5)

  1. 一种用于检测C-KIT基因突变的引物，其特征在于，所述引物如SEQ ID No.1-SEQ ID No.2所示，或者如SEQ ID No.4-SEQ ID No.5所示。
  2. 一种用于检测C-KIT基因突变的探针，其特征在于，所述探针如SEQ ID No.3所示，或如SEQ ID No.6所示。
  3. 一种用于检测C-KIT基因突变的试剂盒，其特征在于，所述试剂盒包括SEQ ID No.1-SEQ ID No.2所示的引物和SEQ ID No.3所示的探针，或者包括SEQ ID No.4-SEQ ID No.5所示的引物和SEQ ID No.6所示的探针。
  4. 如权利要求3所述的试剂盒，其特征在于，其还包括内参基因和内控基因。
  5. 一种检测C-KIT基因突变的方法，其包括以下步骤：
    (1)提取样本中的基因组DNA；
    (2)用权利要求3所述的试剂盒对DNA进行扩增；
    (3)根据荧光PCR仪显示的Ct值判断检测结果。
PCT/CN2016/000352 2015-06-29 2016-06-27 用于检测c-kit基因突变的引物、探针及试剂盒 WO2017000534A1 (zh)

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