CN117025766B - 一种人类alk-e13;a20融合基因检测用dna标准品及其制备方法、应用 - Google Patents
一种人类alk-e13;a20融合基因检测用dna标准品及其制备方法、应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及一种人类ALK‑E13;A20融合基因检测用DNA标准品及其制备方法、应用。本发明所制备的DNA标准品具体为:ALK(E13‑A20)‑1%ALK(E13‑A20)‑2%ALK(E13‑A20)‑5%。本发明制备的人类ALK融合基因突变检测用DNA标准品产品,产品标准品准确度高、均匀度良好。本发明首次建立了人类ALK融合基因突变检测用DNA标准品产品;本发明中ALK(E13;A20)融合基因突变检测定量标准品,量值准确,适用性好。可用于数字PCR或其他方法的验证和性能评估。
Description
技术领域
本发明属于生物技术领域,具体涉及一种人类ALK-E13;A20融合基因检测用DNA标准品及其制备方法、应用。
背景技术
间变性淋巴瘤激酶(ALK)融合基因型肺癌已成为继表皮生长因子(EGFR)突变型肺癌之后的又一个肺癌分子亚型,具有明确的分子靶点、靶点检测技术及上市的靶向药物。
间变性淋巴瘤激酶(ALK)是最新的酪氨酸激酶靶点之一,在大约5%的NSCLC患者中被发现。ALK重排的阳性率大约为3~5%,在腺癌、从未吸烟或少量吸烟的患者中EML4-ALK融合的几率更高一种。ALK是一种融合基因,属于跨膜受体酪氨酸激酶。EML4-ALK融合基因定位于2号染色体的短臂上(2p21和2p23),其5’端为EML4的片段,3’端为ALK的片段,由倒置后的EML4基因片段与残余的ALK片段连接。EML4-ALK的信号转导通路为PI3-K/Akt、STAT3/5、Ras-MEK和PLC-γ/PIP2等,这些通路与细胞存活、增殖和迁移密切相关。它已被证明在体内和体外都参与了肿瘤的发生和发展。在NSCLC中,EML4与ALK基因的融合点不同,有超过15种变异类型,其中V1(EML4 exon 13;ALK exon 20-E13;A20)和V3a/V3b(EML4 exon6ins33;ALK exon 20)是最常见的融合形式。EML4-ALK基因融合可促使ALK基因引起致癌融合蛋白的表达,引起基因表达和信号的激活和失调,进而促使表达这些蛋白的肿瘤细胞增殖和存活。
2011年,美国食品和药品管理局(FDA)批准克唑替尼用于治疗间变型淋巴瘤激酶(ALK)基因重排的非小细胞肺癌。克唑替尼是一种小分子酪氨酸激酶受体抑制剂,靶向分子包括ALK、肝细胞生长因子受体(HGFR,c-Met)和ROS1。EML4-ALK融合基因阳性患者不能从EGFR-TKI的基础治疗中受益,表现为原发耐药。而针对EML4-ALK融合基因阳性的患者,使用克唑替尼等针对ALK基因的小分子抑制剂可以获得良好的临床治疗效果。因此在使用针对ALK基因的小分子抑制剂前,需进行EML4-ALK融合基因突变的检测。
体外诊断试剂作为预防、诊断、治疗监测、预后观察、健康状态评价以及遗传性疾病预测的重要工具,其质量控制非常重要。体外诊断试剂标准物质对于控制体外诊断试剂产品质量、保证临床检测结果准确度,起着十分关键的作用。由于存在很多制约因素,现阶段诊断试剂国家标准物质供应尚不能完全满足注册、生产和质量控制的需要。
发明内容
为了解决以上问题,本发明的目的是提供一种人类ALK-E13;A20融合基因突变检测用DNA标准品产品,该产品标准品准确度高、均匀度良好。本发明首次建立了人类ALK融合基因突变检测用DNA标准品产品。
本发明还提供了人类ALK-E13;A20融合基因突变检测用DNA标准品产品的制备方法。
本发明为了实现上述目的所采用的技术方案为:
本发明提供了一种人类ALK(E13;A20)融合基因突变检测用DNA标准品,所述ALK(E13;A20)融合基因的序列(如SEQ ID NO.1所示)为:
TGACTTGCTTCTTTCACTTAGTTTTTTTTGTTTTGTTTTGTTTGTTTGTTTTTTGAGATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGTGGTCTGATTTTTAGCTTTGCATTTACTTTAAATCATGCTTCAATTAAAGACACACCTTCTTTAATCATTTTATTAGTATTTC。
本发明所制备的人类ALK(E13;A20)融合基因突变检测用DNA标准品,具体为:
本发明制备的上述人类ALK(E13;A20)融合基因突变检测用DNA标准品中:具体的,
所述NCIH3122 gDNA的基因序列(如SEQ ID NO.2所示)为:
ATATAAATGGAGTCATACAATGTGTGGTCTTTTATGACTTGCTTCTTTCACTTAGTTTTTTTTGTTTTGTTTTGTTTGTTTGTTTTTTGAGATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGTGGTCTGATTTTTAGCTTTGCATTTACTTTAAATCATGCTTCAATTAAAGACACACCTTCTTTAATCATTTTATTAGTATTTCTAAGTATGATGGAAAGGTTCAGAGCTCAGGGGAGGATAT;
所述HEK293 gDNA的序列(如SEQ ID NO.3所示)为:GTGTGGTCTTTTATGACTTGCTTCTTTCACTTAGTTTTTTTTGTTTTGTTTTGTTTGTTTGTTTTTTGAGATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGTGGTGCGATTTCGGCTCACTGAACCTCCGCCTCCCAGGTTCAAGCGATTCTC。
进一步的,所述标准品测定用引物探针组为:
检测位点上游引物(如SEQ ID NO.4所示):
5’TTGAGATGGAGTTTCACTCTTGTTG 3’
检测位点下游引物(如SEQ ID NO.5所示):
5’AAGGTGTGTCTTTAATTGAAGCATG 3’
检测位点探针-FAM(如SEQ ID NO.6所示):
5’AGGCTGGAGTGCAGTGGTCTGATT 3’-BHQ1
内参上游引物(如SEQ ID NO.7所示):
5’AACCACCAGAACATTGTTCGC 3’
内参下游引物(如SEQ ID NO.8所示):
5’GTCTCTCGGAGGAAGGACTTGA 3’
内参探针-VIC(如SEQ ID NO.9所示):
5’GCATTGGGGTGAGCCTGCAAT 3’-BHQ1。
本发明还提供了一种人类ALK(E13;A20)融合基因突变检测用DNA标准品的制备方法,包括以下步骤:
(1)提取含有ALK(E13-A20)融合基因的NCIH3122细胞系基因组DNA以及野生型HEK293细胞系基因组DNA作为模板,通过Sanger测序对ALK(E13-A20)基因融合位点和野生型细胞系基因组DNA进行测序验证;
(2)对EML4-AKL融合位置碱基序列进行分析,设计引物探针;
(3)采用数字PCR的方法对NCIH3122细胞系DNA和HEK293野生型细胞系DNA的ALK(E13;A20)融合基因突变频率进行测定,分别重复3次,取3次测定的平均值,作为NCIH3122细胞系的突变频率;
(4)按照不同比例将NCIH3122细胞系DNA和HEK293野生型细胞系DNA进行混合,分别获得1%、2%和5%突变频率的ALK(E13;A20)融合基因DNA标准品,采用数字PCR法进行基因突变频率验证,以及进行标准品均匀性验证。
进一步的,所述数字PCR的扩增体系为:
反应试剂 | 规格 | 用量 |
2XDroplet PCR Supermix for Probes(No dUTP) | 2X | 10μL |
检测位点上游引物 | 20μM | 0.8μL |
检测位点下游引物 | 20μM | 0.8μL |
检测位点探针-FAM | 10μM | 0.5μL |
内参上游引物 | 20μM | 0.8μL |
内参下游引物 | 20μM | 0.8μL |
内参探针-VIC | 10μM | 0.5μL |
DNA模板 | - | 25ng |
dd H2O | - | Up to 20μL |
本发明的有益效果为:
(1)本发明制备的人类ALK融合基因突变检测用DNA标准品产品,产品标准品准确度高、均匀度良好。本发明首次建立了人类ALK融合基因突变检测用DNA标准品产品;
(2)本发明中ALK(E13;A20)融合基因突变检测定量标准品,量值准确,适用性好。可用于数字PCR或其他方法的验证和性能评估。
附图说明
图1为显示数字PCR方法测定HEK293野生型细胞系ALK(E13;A20)融合基因突变频率二维散点图;
图2为显示数字PCR方法测定NCIH3122细胞ALK(E13;A20)融合基因突变频率二维散点图;
图3为显示数字PCR方法测定配制标准品ALK(E13;A20)-1%融合基因突变频率二维散点图;
图4为显示数字PCR方法测定配制标准品ALK(E13;A20)-2%融合基因突变频率二维散点图;
图5为显示数字PCR方法测定配制标准品ALK(E13;A20)-5%融合基因突变频率二维散点图。
具体实施方式
下面通过具体的实施例对本发明的技术方案作进一步的解释和说明。
实施例1
1、细胞系和培养基
NCIH3122细胞系购自上海誉驰生物科技有限,所需的培养基为1640+10%FBS,5%CO2,37℃培养。HEK293野生型细胞系购自湖南丰晖生物科技有限公司,培养基为MEM+10%FBS,5%CO2,37℃培养。细胞培养两周后,收集细胞,提取细胞gDNA作为标准品配制原材料。
2、DNA提取以及Sanger测序
采用康为世纪公司的基因组纯化试剂盒提取细胞系的基因组DNA,采用NanoDrop法微量紫外分光光度计测定基因组在260nm、280nm处的吸光值,OD260/OD280在1.7~1.9之间,DNA纯度符合要求。Qubit荧光计进行细胞基因组DNA浓度测定,分别重复三次,取平均数作为细胞基因组DNA的最终浓度。基因组定量结果见表1,基因组纯度验证见表2。采用Sanger测序对基因组DNA进行验证,测序导出序列如下:
NCIH3122细胞系DNA的ALK(E13-A20)融合基因测序序列如下(未划线部分为EML4基因片段,下划线部分为ALK基因片段):
ATATAAATGGAGTCATACAATGTGTGGTCTTTTATGACTTGCTTCTTTCACTTAGTTTTTTTTGTTTTGTTTTGTTTGTTTGTTTTTTGAGATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGTGGTCTGATTTTTAGCTT TGCATTTACTTTAAATCATGCTTCAATTAAAGACACACCTTCTTTAATCATTTTATTAGTATTTCTAAGTATGATGG AAAGGTTCAGAGCTCAGGGGAGGATAT
HEK293野生型细胞系DNA的EML4基因测序序列如下:(下划线部分为EML4基因融合片段)
GTGTGGTCTTTTATGACTTGCTTCTTTCACTTAGTTTTTTTTGTTTTGTTTTGTTTGTTTGTTTTTTGA GATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGTGGTGCGATTTCGGCTCACTGAACCTCCGCCTCCCAGGTTCAAGCGATTCTC
通过序列比对可得出,NCIH3122细胞系含有ALK(E13-A20)融合基因,同时HEK293野生型细胞系无ALK(E13-A20)融合基因。
表1基因组定量结果
表2基因组纯度验证
根据测定结果NCIH3122细胞系DNA和HEK293细胞系DNA的OD260/OD280在1.7~1.9之间,因此DNA纯度符合要求。
3、对EML4-AK融合位置碱基序列进行分析,进行引物探针设计,利用PrimerPremier5软件设计,可以在正向链或者反向链设计,根据突变位点附近的SNP进行选择,保证引物探针内不含有高频的SNP位点。
ALK(E13-A20)融合基因序列(未划线部分为EML4基因片段,下划线部分为ALK基因片段)
TGACTTGCTTCTTTCACTTAGTTTTTTTTGTTTTGTTTTGTTTGTTTGTTTTTTGAGATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGTGGTCTGATTTTTAGCTTTGCATTTACTTTAAATCATGCTTCAATTAAAGAC ACACCTTCTTTAATCATTTTATTAGTATTTC
检测位点上游引物:5’TTGAGATGGAGTTTCACTCTTGTTG 3’
检测位点下游引物:5’AAGGTGTGTCTTTAATTGAAGCATG 3’
检测位点探针-FAM:5’AGGCTGGAGTGCAGTGGTCTGATT 3’-BHQ1
内参上游引物:5’AACCACCAGAACATTGTTCGC 3’
内参下游引物:5’GTCTCTCGGAGGAAGGACTTGA 3’
内参探针-VIC:5’GCATTGGGGTGAGCCTGCAAT 3’-BHQ1
4、采用数字PCR方法测定NCIH3122细胞系DNA和HEK293野生型细胞系DNA的ALK(E13-A20)融合基因的突变频率,扩增体系如表1。首先将配制好的含有DNA模板的PCR反应液20μL加入到微滴发生卡标记“Sample”的孔内,再向标记“Oil”的孔内加入70μL微滴生成油,然后放入微滴发生器内生成微滴。微滴生成后转移至96孔板内,用加热仪将96孔板的封板膜封好,放置在普通PCR仪上进行扩增。扩增条件为:95℃,3min;40个循环包括95℃,30s,60℃,90s;98℃,5min。
表3 ALK(E13-A20)融合基因数字PCR扩增体系
反应试剂 | 规格 | 用量 |
2XDroplet PCR Supermix for Probes(No dUTP) | 2X | 10μL |
检测位点上游引物 | 20μM | 0.8μL |
检测位点下游引物 | 20μM | 0.8μL |
检测位点探针-FAM | 10μM | 0.5μL |
内参上游引物 | 20μM | 0.8μL |
内参下游引物 | 20μM | 0.8μL |
内参探针-VIC | 10μM | 0.5μL |
DNA模板 | - | 25ng |
dd H2O | - | Up to 20μL |
。
PCR扩增结束后进行微滴的读取和数据分析。采用QuantaSoft软件进行数据的分析处理。
5、采用数字PCR的方法对NCIH3122细胞系DNA和HEK293野生型细胞系DNA的ALK(E13-A20)融合基因突变频率进行测定,分别重复3次,取3次测定的平均值,作为最终的突变频率。数字PCR测定突变频率二维散点图如图1-2,HEK293野生型细胞系DNA没有检测到FAM标记阳性的微滴,标明HEK293野生型细胞系的DNA中不含ALK(E13-A20)融合基因;NCIH3122细胞系DNA测定3个重复,测定结果如表2,通过计算得出ALK(E13-A20)融合基因突变频率为24.20%。
表4数字PCR测定结果
6、配制ALK(E13-A20)融合基因突变的定量标准品
根据ALK(E13-A20)融合基因突变频率进行定量标准品配制,如表5。
表5 ALK(E13-A20)融合基因突变的定量DNA标准品配制表
7、采用数字PCR方法验证不同ALK(E13-A20)融合基因的定量DNA标准品的突变频率,数字PCR测定突变频率二维散点图如图3-5,ALK(E13-A20)-1%、ALK(E13-A20)-2%、ALK(E13-A20)-5%三个标准品突变频率可接受波动范围如表6。采用数字PCR方法分别重复3次,取3次测定结果的平均值,作为DNA标准品的最终突变频率,测定结果如表7
表6 ALK(E13-A20)标准品突变频率可接受波动范围
标准品 | 基因突变频率 | 可接受波动范围 |
ALK(E13-A20)-1% | 1% | 0.8%~1.2% |
ALK(E13-A20)-2% | 2% | 1.6%~2.4% |
ALK(E13-A20)-5% | 5% | 4%~5% |
表7标准品突变频率测定
本发明中ALK(E13-A20)融合基因突变检测定量标准品,量值准确,适用性好。可用于数字PCR或其他方法的验证和性能评估。
Claims (1)
1.一种人类ALK(E13-A20)融合基因突变检测用DNA标准品的制备方法,其特征在于,包括以下步骤:
(1)提取含有ALK(E13-A20)融合基因的NCIH3122细胞系gDNA以及HEK293野生型细胞系gDNA作为模板,通过Sanger测序对ALK(E13-A20)基因融合位点和野生型细胞系gDNA进行测序验证;
(2)对EML4-AKL融合位置碱基序列进行分析,设计引物探针;
(3)采用数字PCR的方法对NCIH3122细胞系gDNA和HEK293野生型细胞系gDNA的ALK(E13-A20)融合基因突变频率进行测定,分别重复3次,取3次测定的平均值,作为NCIH3122细胞系的突变频率;
(4)按照不同比例将NCIH3122细胞系gDNA和HEK293野生型细胞系gDNA进行混合,分别获得1%、2%和5%突变频率的ALK(E13-A20)融合基因DNA标准品,采用数字PCR法进行基因突变频率验证,以及进行标准品均匀性验证;
所述ALK(E13-A20)融合基因的序列为:
TGACTTGCTTCTTTCACTTAGTTTTTTTTGTTTTGTTTTGTTTGTTTGTTTTTTGAGATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGTGGTCTGATTTTTAGCTTTGCATTTACTTTAAATCATGCTTCAATTAAAGACACACCTTCTTTAATCATTTTATTAGTATTTC;
所述NCIH3122细胞系gDNA的ALK( E13-A20)融合基因序列为:
ATATAAATGGAGTCATACAATGTGTGGTCTTTTATGACTTGCTTCTTTCACTTAGTTTTTTTTGTTTTGTTTTGTTTGTTTGTTTTTTGAGATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGTGGTCTGATTTTTAGCTTTGCATTTACTTTAAATCATGCTTCAATTAAAGACACACCTTCTTTAATCATTTTATTAGTATTTCTAAGTATGATGGAAAGGTTCAGAGCTCAGGGGAGGATAT;
所述HEK293 野生型细胞系gDNA的EML4基因序列为: GTGTGGTCTTTTATGACTTGCTTCTTTCACTTAGTTTTTTTTGTTTTGTTTTGTTTGTTTGTTTTTTGAGATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGTGGTGCGATTTCGGCTCACTGAACCTCCGCCTCCCAGGTTCAAGCGATTCTC;
所述DNA标准品测定用引物探针组为:
检测位点上游引物:5’ TTGAGATGGAGTTTCACTCTTGTTG 3’
检测位点下游引物:5’ AAGGTGTGTCTTTAATTGAAGCATG 3’
检测位点探针-FAM:5’ AGGCTGGAGTGCAGTGGTCTGATT 3’-BHQ1
内参上游引物:5’ AACCACCAGAACATTGTTCGC 3’
内参下游引物:5’ GTCTCTCGGAGGAAGGACTTGA 3’
内参探针-VIC:5’ GCATTGGGGTGAGCCTGCAAT 3’-BHQ1;
所述数字PCR的扩增体系为:
所述数字PCR的扩增条件为:95℃,3min;40个循环包括95℃,30s,60℃,90s;98℃,5min。
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