CN112430648A - 检测样本中etv6-runx1融合基因的寡核苷酸、方法和试剂盒 - Google Patents
检测样本中etv6-runx1融合基因的寡核苷酸、方法和试剂盒 Download PDFInfo
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Abstract
本发明涉及检测样本中ETV6‑RUNX1融合基因的寡核苷酸、方法和试剂盒,主要包括检测ETV6‑RUNX1融合基因的上游引物ETV6‑F、下游引物RUNX1‑R和探针ETV6‑Probe以及检测ABI内参基因的上游引物ABL‑F、下游引物ABL‑R和探针ABL‑Probe,用于检测急性B淋巴细胞白血病患者体内ETV6‑RUNX1融合基因情况,便于开展相关辅助诊断及治疗。
Description
技术领域
本发明属于生命科学和生物技术领域,特别设计提供检测样本中ETV6-RUNX1融合基因的寡核苷酸、方法和试剂盒,采用探针实时荧光PCR技术,能够对人类B-ALL中的ETV6-RUNX1融合基因进行检测。
背景技术
白血病的发生是一个复杂而多步骤的过程,染色体突变可形成具有肿瘤特性的融合基因,也可使一些在细胞生长、凋亡调控过程中起重要作用的基因表达失控,从而干扰细胞增殖、分化、成熟和凋亡的正常调节途径,最终导致白血病发生。白血病是儿童期最常见的恶性肿瘤性疾病,发病率3-5/10万,也是严重威胁儿童健康的疾病。儿童白血病以急性白血病为主,其中伴t(12;21)(p13;q22)细胞遗传学异常的急性淋巴细胞白血病(ALL)占儿童ALL的20%~25%。检测细胞及分子遗传学改变对白血病诊断、制订合理的治疗方案及预后判断有重要的指导意义。例如t(12;21)及其形成的ETV6/RUNX1基因多见于儿童患者,且这些患者对化疗敏感,可采用较温和的化疗方案也能达到较好疗效。
t(12;21)(p13;q22)最早在1995年被报道,这种易位导致位于染色体12p13的TEL基因和位于染色体21q22的AML1基因断裂,重接形成TEL-AML1融合基因。在美国国家综合癌症网络(NCCN)指南(2015.V2)中,将TEL-AMLl更名为ETV6-RUNX1。但这种易位使用常规的染色体核型分析几乎不能够被检出,只能用RT-PCR和FISH方法检出。经过研究表明,PCR技术是检测这个融合基因的灵敏且可靠的方法。
ETV6基因属ETS转录因子家族成员,编码转录抑制因子。ETS家族成员在细胞的增值、分化、迁移、组织重塑、血管生成及造血细胞转化过程中都起着重要的作用。ETV6基因定位于12p13,DNA全长约300kb,共8个外显子,编码452个氨基酸。ETS家族成员的羧基端都有一个ETS结构域,该结构域是由85个氨基酸组成的高度保守区域,可识别核心元件GGAA从而介导ETV6与DNA相结合,而在其氨基端同样有一高度保守的HLH结构域(又称B结构域或point结构域),由652个氨基酸组成,它可以介导ETV6基因之间或与其它不同转录因子之间的蛋白-蛋白相互作用形成同二聚体或异二聚体,从而参与基因的表达和调控。
ETV6/RUNX1融合基因可以使天冬酰胺酸合成酶水平降低,故此类患儿常对左旋门冬酰胺敏感,化疗效果佳,它的表达是独立的预后较好的指标之一,这些病人可以应用较弱的化疗方案,以减轻化疗的副作用但不降低无病生存率。
ETV6/RUNX1融合基因是儿童B-ALL中预后较好的生物学标志,但是,研究者也发现其另一个明显的临床特点,即在ETV6/RUNX1阳性的B-ALL患儿中,总有部分患儿会发生复发,且多是在维持期或停药后3年以上的晚期或远期复发,提示该类型儿童白血病肿瘤细胞的恶性度虽然低于阴性患儿,但仍有部分患儿难以通过现有化疗方案以彻底清除残存的白血病细胞,故由此可能成为日后复发的根源。
因此,如能在疾病诊治初期就能对可能发生的复发作出早期预警,并由此制定针对该类型患儿的个体化治疗方案,以及在复发后对复发的白血病细胞克隆来源做出精准的鉴定,将有助于提高患儿的远期疗效。
另外,免疫残留作为B细胞ALL微小残留病(MRD)监测指标的临床意义已获得肯定。有文献指出,ETV6-RUNXl融合基因和免疫残留存在一定的相关性,且融合基因阳性早于免疫残留,说明该融合基因可作为该类型白血病患者移植后MRD监测更为敏感的指标。ETV6-RUNXl融合基因可作为移植后MRD的监测指标,并具有更高的敏感性和特异性。
ETV6-RUNXl融合基因检测的常用技术有荧光原位杂交技术(FISH)、实时定量PCR(RQ-PCR)等方法。FISH检测结果较为直观,但是试验过程繁琐,涉及试剂种类繁多,费时费力,且结果需经验丰富的专业人士来判读,结果判读存在较大的主观性。RQ-PCR采用TaqMan探针荧光定量技术,综合生物学、酶学和荧光化学于一体,从扩增到结果分析均在PCR反应管封闭状态下进行,解决了PCR产物污染而导致假阳性的问题,同时也提高了敏感度,其结果用拷贝数表示,实现了对PCR产物的准确定量,易于统一标准,与定性PCR技术相比,具有特异度好,灵敏度高,线性关系好,操作简单,自动化程度高、防污染,有较大的线性范围等优点。能够满足ETV6-RUNXl基因重排的检测,被认为是目前首选检测方法,用于评价治疗效果、预测预后。实时荧光定量PCR中常见的方法有SYBR Green I染料法,双探针杂交法以及TaqMan技术等。其中SYBR Green I由于是非饱和染料,特异性不如双探针杂交法以及TaqMan法,必须通过观察溶解曲线来判断其特异性;而双探针杂交法成本又较为昂贵。因此本研究采用实时荧光PCR技术结合TaqMan探针法应用于ETV6-RUNXl的基因重排检测。
发明内容
本发明设计了检测内参/目的基因用引物、探针序列,用实时荧光PCR技术检测ETV6-RUNXl基因重排。通过调整引物、探针浓度及比例,优化PCR的反应体系和反应条件,使扩增效率和速率均达到最佳。
本发明提供了用于检测样本中ETV6-RUNX1融合基因的寡核苷酸,所述寡核苷酸包括检测ETV6-RUNX1融合基因的上游引物ETV6-F、下游引物RUNX1-R和探针ETV6-Probe,其碱基序列为:
ETV6-F:CTCTGTCTCCCCGCCTGAA
RUNX1-R:GCACCGACAGCCCCAACTT
ETV6-Probe:FAM-CACGCCATGCCCATTGGGA-TAMRA。
进一步地,ETV6-F:RUNX1-R:ETV6-Probe的摩尔比为1:1:1。
进一步地,所述寡核苷酸还包括检测ABL内参基因的上游引物ABL-F、下游引物ABL-R和探针ABL-Probe,其碱基序列为:
ABL-F:GATACGAAGGGAGGGTGTACCA
ABL-R:CTCGGCCAGGGTGTTGAA
ABL-Probe:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA。
进一步地,ABL-F:ABL-R:ABL-Probe的摩尔比为1:1:1。
本发明还提供了一种检测样本中ETV6-RUNX1融合基因的方法,包括以下步骤:
(1)提取样本中的RNA;
(2)将(1)提取出的RNA逆转录为cDNA;
(3)加入(2)中所述的cDNA到反应管中,利用检测ETV6-RUNX1融合基因的上游引物ETV6-F、下游引物RUNX1-R和探针ETV6-Probe检测样本中的ETV6-RUNX1融合基因荧光信号;利用检测ABL内参基因的上游引物ABL-F、下游引物ABL-R和探针ABL-Probe检测样本中的ABL内参基因荧光信号,其中
ETV6-F:CTCTGTCTCCCCGCCTGAA
RUNX1-R:GCACCGACAGCCCCAACTT
ETV6-Probe:FAM-CACGCCATGCCCATTGGGA-TAMRA
ABL-F:GATACGAAGGGAGGGTGTACCA
ABL-R:CTCGGCCAGGGTGTTGAA
ABL-Probe:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA
(4)根据(3)中的ETV6-RUNX1融合基因荧光信号和ABL内参基因荧光信号,确定样本中的ETV6-RUNX1融合基因相对表达量。
本发明还提供了一种检测样本中ETV6-RUNX1融合基因的试剂盒,所述试剂盒包括检测体系PCR反应液,所述检测体系PCR反应液包括检测ETV6-RUNX1融合基因的上游引物ETV6-F、下游引物RUNX1-R和探针ETV6-Probe以及检测ABL内参基因的上游引物ABL-F、下游引物ABL-R和探针ABL-Probe,其中,
ETV6-F:CTCTGTCTCCCCGCCTGAA
RUNX1-R:GCACCGACAGCCCCAACTT
ETV6-Probe:FAM-CACGCCATGCCCATTGGGA-TAMRA
ABL-F:GATACGAAGGGAGGGTGTACCA
ABL-R:CTCGGCCAGGGTGTTGAA
ABL-Probe:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA。
进一步地,ETV6-F:RUNX1-R:ETV6-Probe的摩尔比为1:1:1。
进一步地,ABL-F:ABL-R:ABL-Probe的摩尔比为1:1:1。
进一步地,所述试剂盒还包括阳性对照品、阴性对照品和空白对照品,所述阳性对照品为含有ETV6-RUNX1 cDNA序列的质粒溶液,所述阴性对照品为不含有ETV6-RUNX1 cDNA序列的质粒溶液,所述空白对照品为生理盐水或不加任何物质。
进一步地,所述试剂盒还包括样本RNA提取液和红细胞裂解液,所述红细胞裂解液包括16μmol/L氯化铵、1mmol/L碳酸氢钾和12.5μmol/L EDTA,所述样本RNA提取液包括TRIzol、氯仿、异丙醇、75%乙醇和RNase-free水。
本发明的有益效果:本发明将实时荧光PCR技术结合采用Tapman探针,利用双标准曲线的方法,分别构建内参基因ABL和ETV6-RUNXl目的基因的定量标准曲线,检测受测者体内ETV6-RUNXl的表达水平。相比于以往的FISH和△△CT法,该方法具有精度高,结果便于判读等优点。加之该方法将反应体系所需的引物、探针进行合理配比和优化,使实验条件达到最佳,从而省去了繁琐的条件摸索环节,大大提升了实验效率。该方法经测试特异性好,灵敏度高,操作简便。有助于临床上B-ALL患者体内ETV6-RUNXl融合基因的检测,对于白血病分型诊断、调整治疗方案、评价治疗效果、预测预后、预防临床复发都具有重要意义。
附图说明
图1是使用本方法检测阳性质粒的结果图。
图2是灵敏度检测的结果图。
图3是使用本方法检测临床样本1的结果图。
具体实施方式
实施例1
本发明用于辅助临床上B-ALL的分型诊断及个体化治疗方案制定的方法。主要包括以下试剂:红细胞裂解液,其配方为:16μmol/L氯化铵、1mmol/L碳酸氢钾和12.5μmol/LEDTA;样本RNA提取液:TRIzol、氯仿、异丙醇、75%乙醇和RNase-free水;检测体系PCR反应液:ReverTra Ace qPCR RT Kit(TOYOBO公司);THUNDERBIRD Probe qPCR Mix(2×)、ABL内参基因的引物ABL-F、ABL-R和探针ABL-Probe以及ETV6-RUNXl目的基因的引物ETV6-F、RUNX1-R和探针ETV6-probe均为10μM;
其中检测内参基因ABL和目的基因ETV6-RUNXl的引物和探针,如表1所述:
表1.引物和探针序列
阳性对照品:含有ETV6-RUNX1 cDNA序列的质粒溶液(即含有ETV6-RUNX1 cDNA序列的阳性质粒);
阴性对照品:不含有ETV6-RUNX1 cDNA序列的质粒溶液;
空白对照品:生理盐水或不加任何物质。
如图1所示,含有ETV6-RUNX1 cDNA序列的阳性质粒和ABL内参基因均表达。
实施例2
本发明方法的操作流程:
(1)抽提血液中的总RNA:在洁净的1.5ml的离心管中加入1ml红细胞裂解液,取抗凝血0.5ml混匀。室温静置10min;1500rpm离心5min,弃上清,收集底部的细胞;再次加入0.5ml红细胞裂解液,1500rpm离心5min,弃上清,收集底部的细胞;向细胞中加入1mlTRIzol,反复吹打直至沉淀完全溶解,室温静止5min;加入0.2ml氯仿,震荡均匀;14000rpm4℃离心10min,吸取上清层转移至另一新的离心管中;加入等体积的异丙醇,上下充分混匀,室温静置10min;14000rpm 4℃离心10min,弃上清,加入75%乙醇1ml,轻轻上下颠倒洗涤管壁;14000rpm 4℃离心5min,弃乙醇;室温干燥10-15min,加入20ulRNase-free水溶解沉淀。
(2)参考TOYOBO公司的Rever Tra Ace qPCR RT Kit试剂盒说明书,将RNA反转为cDNA。
(3)试剂配置:按检测人份数配置检测体系PCR反应液各XμL,每人份23μL分装:
X=23μL反应液×(8份内参(标准曲线)+8份目的基因(标准曲线)+n份样本+1份阳性对照+1份阴性对照+1份空白对照);
(4)加样:加入检测体系PCR反应液中2μL cDNA;阳性对照和阴性对照直接加2μL阳性对照品(含有ETV6-RUNX1 cDNA序列的阳性质粒)和阴性对照品;空白对照加2μL生理盐水或不加任何物质。
(5)检测:检测在实时荧光PCR仪上进行,可用仪器包括ABI7300,7500(美国Applied Biosystems公司)等。反应条件:95℃预变性1min;95℃15s,58℃35sec 40个循环,荧光信号于58℃40sec时采集。
(6)结果判断:将阈值线调整至背景信号及阴性扩增线以上,系统根据标准曲线和Ct值自动计算出拷贝数。
1)内参阳性时,检测结果才认为有效;
2)阳性判断标准:Ct<35,为阳性;35≤Ct≤37,为疑似阳性,需要再次验证;Ct>37,为阴性。
实施例3
用本发明核酸检测方法检测灵敏度:
将ETV6-RUNX1阳性质粒按照拷贝数计算公式计算出原始拷贝数,在此基础上设计若干稀释度得到拷贝数为1010、109、108、107、106、105、104、103、102、10、1copies/μl的阳性质粒,以102、10、1拷贝数的质粒作为模板进行PCR扩增,每个浓度重复做10个复孔。结果如表2所示,图2为ETV6-RUNXl阳性质粒灵敏度检测扩增曲线图。由表2可知,100copies/μL的质粒都能全部出现扩增,而10copies/μL的质粒只部分出现扩增因此,本发明对ETV6-RUNX1质粒的检测下限为100copies/μL。
表2 ETV6-RUNX1阳性质粒的灵敏度检测Ct值
实施例4
采用本发明核酸检测方法检测健康体检人群样本
取待检的健康体检样本22例,按实施例2所述方法提取总RNA、配制试剂并检测。
每份样本加入检测体系PCR反应液中2μL。同时做阳性对照、阴性对照和空白对照,内参基因/目的基因的标准曲线各一份。一台96孔的荧光PCR仪可同时检测22份样本,每个样本2次重复,一份阳性对照,一份阴性对照,检测时间仅为70分钟。22例筛查样本中所有样本的ABL均起线,但ETV6-RUNXl未有样本出现起线。结果如表3所示。
表3 22例健康体检样本ETV6-RUNXl表达水平
实施例5
采用本发明核酸检测方法检测临床样本
取待检的临床样本22例,按实施例2所述方法提取基因组、配制试剂并检测。
每份样本加入检测体系PCR反应液中2μL。同时做阳性,阴性,空白对照,内参基因/目的基因的标准曲线各一份。一台96孔的荧光PCR仪可同时检测22份样本,每个样本2次重复,一份阳性对照,一份阴性对照,检测时间仅为70分钟。22例筛查样本中所有样本的ABL均起线,但ETV6-RUNXl未有样本出现起线,即临床样本均为阴性。如图3所示,1号样本为阴性。实验结果如表4所示:
表4 22例临床样本ETV6-RUNXl表达水平
序列表
<110> 福州艾迪康医学检验所有限公司
<120> 检测样本中ETV6-RUNX1融合基因的寡核苷酸、方法和试剂盒
<160> 6
<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)
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ctctgtctcc ccgcctgaa 19
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<213> 人工序列(Artificial Sequence)
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gcaccgacag ccccaactt 19
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<212> DNA
<213> 人工序列(Artificial Sequence)
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cacgccatgc ccattggga 19
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<213> 人工序列(Artificial Sequence)
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gatacgaagg gagggtgtac ca 22
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ctcggccagg gtgttgaa 18
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tgcttctgat ggcaagctct acgtctcct 29
Claims (10)
1.用于检测样本中ETV6-RUNX1融合基因的寡核苷酸,其特征在于,所述寡核苷酸包括检测ETV6-RUNX1融合基因的上游引物ETV6-F、下游引物RUNX1-R和探针ETV6-Probe,其碱基序列为:
ETV6-F:CTCTGTCTCCCCGCCTGAA
RUNX1-R:GCACCGACAGCCCCAACTT
ETV6-Probe:FAM-CACGCCATGCCCATTGGGA-TAMRA。
2.如权利要求1所述的寡核苷酸,其特征在于,ETV6-F:RUNX1-R:ETV6-Probe的摩尔比为1:1:1。
3.如权利要求1所述的寡核苷酸,其特征在于,所述寡核苷酸还包括检测ABL内参基因的上游引物ABL-F、下游引物ABL-R和探针ABL-Probe,其碱基序列为:
ABL-F:GATACGAAGGGAGGGTGTACCA
ABL-R:CTCGGCCAGGGTGTTGAA
ABL-Probe:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA。
4.如权利要求3所述的引物和探针,其特征在于,ABL-F:ABL-R:ABL-Probe的摩尔比为1:1:1。
5.一种检测样本中ETV6-RUNX1融合基因的方法,其特征在于:
(1)提取样本中的RNA;
(2)将(1)提取出的RNA逆转录为cDNA;
(3)加入(2)中所述的cDNA到反应管中,利用检测ETV6-RUNX1融合基因的上游引物ETV6-F、下游引物RUNX1-R和探针ETV6-Probe检测样本中的ETV6-RUNX1融合基因荧光信号;利用检测ABL内参基因的上游引物ABL-F、下游引物ABL-R和探针ABL-Probe检测样本中的ABL内参基因荧光信号,其中
ETV6-F:CTCTGTCTCCCCGCCTGAA
RUNX1-R:GCACCGACAGCCCCAACTT
ETV6-Probe:FAM-CACGCCATGCCCATTGGGA-TAMRA
ABL-F:GATACGAAGGGAGGGTGTACCA
ABL-R:CTCGGCCAGGGTGTTGAA
ABL-Probe:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA
(4)根据(3)中的ETV6-RUNX1融合基因荧光信号和ABL内参基因荧光信号,确定样本中的ETV6-RUNX1融合基因相对表达量。
6.一种检测样本中ETV6-RUNX1融合基因的试剂盒,所述试剂盒包括检测体系PCR反应液,其特征在于,所述检测体系PCR反应液包括检测ETV6-RUNX1融合基因的上游引物ETV6-F、下游引物RUNX1-R和探针ETV6-Probe以及检测ABL内参基因的上游引物ABL-F、下游引物ABL-R和探针ABL-Probe,其中,
ETV6-F:CTCTGTCTCCCCGCCTGAA
RUNX1-R:GCACCGACAGCCCCAACTT
ETV6-Probe:FAM-CACGCCATGCCCATTGGGA-TAMRA
ABL-F:GATACGAAGGGAGGGTGTACCA
ABL-R:CTCGGCCAGGGTGTTGAA
ABL-Probe:FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA。
7.如权利要求6所述的试剂盒,其特征在于,ETV6-F:RUNX1-R:ETV6-Probe的摩尔比为1:1:1。
8.如权利要求6所述的试剂盒,其特征在于,ABL-F:ABL-R:ABL-Probe的摩尔比为1:1:1。
9.如权利要求6所述的试剂盒,所述试剂盒还包括阳性对照品、阴性对照品和空白对照品,其特征在于,所述阳性对照品为含有ETV6-RUNX1 cDNA序列的质粒溶液,所述阴性对照品为不含有ETV6-RUNX1 cDNA序列的质粒溶液,所述空白对照品为生理盐水或不加任何物质。
10.如权利要求6~9之一所述的试剂盒,所述试剂盒还包括样本RNA提取液和红细胞裂解液,其特征在于,所述红细胞裂解液包括16μmol/L氯化铵、1mmol/L碳酸氢钾和12.5μmol/L EDTA,所述样本RNA提取液包括TRIzol、氯仿、异丙醇、75%乙醇和RNase-free水。
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