CN113913520A - 检测人类idh1基因r132h突变的探针法及其试剂盒 - Google Patents
检测人类idh1基因r132h突变的探针法及其试剂盒 Download PDFInfo
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Abstract
本发明公开了检测人类IDH1基因R132H突变的探针法及其试剂盒,本发明涉及生物医疗技术领域,检测人类IDH1基因R132H突变的探针法,包括以下步骤:(1)在IDH1R132HPCR反应混合液中加入人类基因组DNA,形成R132H反应体系并进行荧光定量PCR扩增;(2)结果判断:根据PCR扩增的突变Ct值进行分析;IDH1R132H反应混合液中包括:具有如序列表中SEQIDNo.3所示序列的IDH1R132H前向引物、具有如序列表中SEQIDNo.4所示序列的IDH1R132H反向引物和含有荧光染料的PCR预混液;本发明的有益效果在于:该探针法和试剂盒为临床提供了一种实现对IDH1基因R132H突变的高灵敏度检测,并且IDH1基因R132H突变的检测速度快、效率高、特异性强。
Description
技术领域
本发明涉及生物医疗技术领域,尤其是涉及检测人类IDH1基因R132H突变的探针法及其试剂盒。
背景技术
异柠檬酸脱氢酶(IDH)家族包括以NAD或NADP为辅助因子,催化异柠檬酸的氧化脱羧反应生成α-酮戊二酸的酶类,同时分别生成NADH或NADPH。在哺乳动物细胞中, IDH同工酶有以下三种形式:依赖NAD的线粒体IDH1,依赖NADP的线粒体IDH2,依 赖NADP的胞质IDH3。编码依赖NAD的人IDH1的IDH1基因位于染色体2q33.3,并且 定位于细胞质和过氧化物酶体中;编码依赖NADP的线粒体IDH2酶的IDH2基因位于 染色体15q26.1,但是现在检测人类IDH1基因R132H突变的探针法的灵敏度检测率比较低,并且速度慢同时其特异性不强。
发明内容
本发明旨在克服上述现有技术的至少缺陷,提供检测人类IDH1基因R132H突变的探针法,包括以下步骤:
(1)在IDH1R132HPCR反应混合液中加入人类基因组DNA,形成R132H反应体系并进行荧光定量PCR扩增;
(2)结果判断:根据PCR扩增的突变Ct值进行分析。
作为优选的,所述IDH1R132H反应混合液中包括:具有如序列表中SEQIDNo.3所示序列的IDH1R132H前向引物、具有如序列表中SEQIDNo.4所示序列的IDH1R132H反向引物和含有荧光染料的PCR预混液。
作为优选的,所述在IDH1R132H反应混合液中加入人类基因组DNA:在10μLIDH1R132H反应混合液中加入4.5μL、浓度为9ng/μL的人类基因组DNA,其中:在IDH1R132H反应混合液中IDH1R132H前向引物、IDH1R132H反向引物和含有荧光染料的3×PCR预混液的体积比为(1.5-2):(1.5-2):(7.5-8),优选为1.55:1.55:8;在IDH1R132H反应混合液中IDH1R132H前向引物和IDH1R132H反向引物的浓度均为2-8μM,优选3-4μM,更优选4.5μM。
作为优选的,所述荧光定量PCR方法的扩增过程为:(1)95℃、4min、循环数为1;(2)95℃、18s,68℃、20s,循环数为18;(3)95℃、15s,57.6℃、20s,循环数为26;(4)升温至98℃。
本发明提供根据以上所述的检测人类EGFR基因L858R突变的检测方法,所述对Ct值进行分析的步骤为:
当样品在R132H反应体系的突变Ct值大于或等于阴性临界值19时,则该样品的R132H突变为阴性或小于本试剂盒的检测最低阈值;
当样品在R132H反应体系的突变Ct值小于阴性临界值19时,按照以下标准进行判断:当该样品在R132H反应体系的突变Ct值小于或等于阳性临界值16时,则该样品的R132H突变为阳性,即强阳;当该样品在R132H反应体系的突变Ct值大于阳性临界值16时,则计算R132H反应体系的△Ct,ΔCt值=R132H反应体系突变Ct值-该样本在内标反应体系的Ct值,若反应体系的△Ct值小于相应的△CtCut-off值9,则该样品的R132H突变也为阳性,即弱阳,若反应体系的△Ct值大于或等于相应的△CtCut-off值9,则该样品的R132H突变为阴性或低于本试剂盒的检测最低阈值。
采用上述技术方案,所取得的有益效果是:
该探针法和试剂盒为临床提供了一种实现对IDH1基因R132H突变的高灵敏度检测,并且IDH1基因R132H突变的检测速度快、效率高、特异性强。
具体实施方式
为使本发明的目的、技术方案及优点更加清楚明白,以下结合具体实施方式,对本发明进行进一步的详细说明。应当理解的是,此处所描述的具体实施方式仅用以解释本发明,并不限定本发明的保护范围。
本申请是检测人类IDH1基因R132H突变的探针法及其试剂盒,所述检测人类IDH1基因R132H突变的探针法,包括以下步骤:
(1)在IDH1R132HPCR反应混合液中加入人类基因组DNA,形成R132H反应体系并进行荧光定量PCR扩增,所述IDH1R132H反应混合液中包括:具有如序列表中SEQIDNo.3所示序列的IDH1R132H前向引物、具有如序列表中SEQIDNo.4所示序列的IDH1R132H反向引物和含有荧光染料的PCR预混液,所述在IDH1R132H反应混合液中加入人类基因组DNA:在10μLIDH1R132H反应混合液中加入4.5μL、浓度为9ng/μL的人类基因组DNA,其中:在IDH1R132H反应混合液中IDH1R132H前向引物、IDH1R132H反向引物和含有荧光染料的3×PCR预混液的体积比为(1.5-2):(1.5-2):(7.5-8),优选为1.55:1.55:8;在IDH1R132H反应混合液中IDH1R132H前向引物和IDH1R132H反向引物的浓度均为2-8μM,优选3-4μM,更优选4.5μM,所述荧光定量PCR方法的扩增过程为:(1)95℃、4min、循环数为1;(2)95℃、18s,68℃、20s,循环数为18;(3)95℃、15s,57.6℃、20s,循环数为26;(4)升温至98℃;
(2)结果判断:根据PCR扩增的突变Ct值进行分析。
本发明提供根据以上所述的检测人类EGFR基因L858R突变的检测方法,所述对Ct值进行分析的步骤为:
当样品在R132H反应体系的突变Ct值大于或等于阴性临界值19时,则该样品的R132H突变为阴性或小于本试剂盒的检测最低阈值;
当样品在R132H反应体系的突变Ct值小于阴性临界值19时,按照以下标准进行判断:当该样品在R132H反应体系的突变Ct值小于或等于阳性临界值16时,则该样品的R132H突变为阳性,即强阳;当该样品在R132H反应体系的突变Ct值大于阳性临界值16时,则计算R132H反应体系的△Ct,ΔCt值=R132H反应体系突变Ct值-该样本在内标反应体系的Ct值,若反应体系的△Ct值小于相应的△CtCut-off值9,则该样品的R132H突变也为阳性,即弱阳,若反应体系的△Ct值大于或等于相应的△CtCut-off值9,则该样品的R132H突变为阴性或低于本试剂盒的检测最低阈值。
Claims (5)
1.检测人类IDH1基因R132H突变的探针法,其特征在于,包括以下步骤:
(1)在IDH1R132HPCR反应混合液中加入人类基因组DNA,形成R132H反应体系并进行荧光定量PCR扩增;
(2)结果判断:根据PCR扩增的突变Ct值进行分析。
2.根据权利要求1所述的检测人类IDH1基因R132H突变的试剂盒,其特征在于:所述IDH1R132H反应混合液中包括:具有如序列表中SEQIDNo.3所示序列的IDH1R132H前向引物、具有如序列表中SEQIDNo.4所示序列的IDH1R132H反向引物和含有荧光染料的PCR预混液。
3.根据权利要求1所述的检测人类IDH1基因R132H突变的试剂盒,其特征在于:所述在IDH1R132H反应混合液中加入人类基因组DNA:在10μLIDH1R132H反应混合液中加入4.5μL、浓度为9ng/μL的人类基因组DNA,其中:在IDH1R132H反应混合液中IDH1R132H前向引物、IDH1R132H反向引物和含有荧光染料的3×PCR预混液的体积比为(1.5-2):(1.5-2):(7.5-8),优选为1.55:1.55:8;在IDH1R132H反应混合液中IDH1R132H前向引物和IDH1R132H反向引物的浓度均为2-8μM,优选3-4μM,更优选4.5μM。
4.根据权利要求3所述的检测人类IDH1基因R132H突变的试剂盒,其特征在于:所述荧光定量PCR方法的扩增过程为:(1)95℃、4min、循环数为1;(2)95℃、18s,68℃、20s,循环数为18;(3)95℃、15s,57.6℃、20s,循环数为26;(4)升温至98℃。
5.权利要求4所述的检测人类EGFR基因L858R突变的检测方法,其特征在于,所述对Ct值进行分析的步骤为:
当样品在R132H反应体系的突变Ct值大于或等于阴性临界值19时,则该样品的R132H突变为阴性或小于本试剂盒的检测最低阈值;
当样品在R132H反应体系的突变Ct值小于阴性临界值19时,按照以下标准进行判断:当该样品在R132H反应体系的突变Ct值小于或等于阳性临界值16时,则该样品的R132H突变为阳性,即强阳;当该样品在R132H反应体系的突变Ct值大于阳性临界值16时,则计算R132H反应体系的△Ct,ΔCt值=R132H反应体系突变Ct值-该样本在内标反应体系的Ct值,若反应体系的△Ct值小于相应的△CtCut-off值9,则该样品的R132H突变也为阳性,即弱阳,若反应体系的△Ct值大于或等于相应的△CtCut-off值9,则该样品的R132H突变为阴性或低于本试剂盒的检测最低阈值。
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