实时荧光聚合酶链式反应偶联高特异性核酸侵入反应检测单核苷酸多态性

建立了实时荧光聚合酶链式反应（ PCR）偶联高特性核酸侵入反应检测单核苷酸多态性（ SNP）的方法。优化了体系中flap核酸内切酶1（FEN1酶）和野生型检测探针等用量,确定了最佳反应条件,即FEN1酶用量为1.5 U,野生型检测探针用量为0.125μmol/L,0.5μmol/L Invader突变型检测探针,各0.25μmol/L通用野生型（ VIC）和突变型（ FAM）荧光共振转移发卡探针,显著降低了野生型样本和突变型样本背景信号,避免了背景信号对检测结果分型的干扰。采用本方法对编码乙醛脱氢酶2（ ALDH2）基因ALDH2＊2位点21例样本、细胞色素P4502C19基因CYP2C19＊...

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Bibliographic Details
Published in分析化学 Vol. 43; no. 7; pp. 1001 - 1008
Main Author 郑梦琳 齐谢敏 童欢 刘云龙 邹秉杰 宋沁馨 周国华
Format Journal Article
LanguageChinese
Published 药物质量与安全预警教育部重点实验室，中国药科大学，南京210009 2015
南京军区南京总医院药理科，南京210002%南京军区南京总医院药理科,南京,210002
Subjects
2C19基因
乙醛脱氢酶2基因
单核苷酸多态性
核酸侵入反应
细胞色素P450
乙醛脱氢酶2基因
核酸侵入反应
单核苷酸多态性
Invader assay
实时荧光PCR
细胞色素P450 2C19基因
Single nucleotide polymorphism
Cytochrome p450 2 C192 gene
Real time polymerase chain reaction
Aldehyde dehydrogenase-2 gene
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ISSN0253-3820
DOI10.11895/j.issn.0253-3820.150051

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Summary:建立了实时荧光聚合酶链式反应（ PCR）偶联高特性核酸侵入反应检测单核苷酸多态性（ SNP）的方法。优化了体系中flap核酸内切酶1（FEN1酶）和野生型检测探针等用量,确定了最佳反应条件,即FEN1酶用量为1.5 U,野生型检测探针用量为0.125μmol/L,0.5μmol/L Invader突变型检测探针,各0.25μmol/L通用野生型（ VIC）和突变型（ FAM）荧光共振转移发卡探针,显著降低了野生型样本和突变型样本背景信号,避免了背景信号对检测结果分型的干扰。采用本方法对编码乙醛脱氢酶2（ ALDH2）基因ALDH2＊2位点21例样本、细胞色素P4502C19基因CYP2C19＊2和CYP2C19＊3位点各19例样本进行分型检测,结果表明, AL-DH2＊2位点GG纯合10例,GA杂合8例,AA纯合3例；CYP2C19＊2位点GG纯合9例,GA杂合8例,AA纯合2例；CYP2C19＊3位点GG纯合18例,GA杂合1例。使用焦磷酸测序进行验证,两种方法检测结果一致。本方法特异性好、操作简便、耗时短、成本低,可实现对SNP单管闭管无污染的分型检测。
Bibliography:Real time polymerase chain reaction;Invader assay;Single nucleotide polymorphism;Aldehyde dehydrogenase-2 gene;Cytochrome p450 2 C19＊2 gene
22-1125/O6
A method for the real-time polymerase chain reaction （ PCR ） coupled with high specific invader assay to detect single nucleotide polymorphism （ SNP） was established. To reduce the background signal, the amount of flap endonuclease 1 （ FEN1 enzyme ） and wild-type detection probe was optimized. Under the optimum conditions including 0. 05 μmo/L invasive oligonucleotide probe, 0. 125 μmol/L wild-type detection probe, 0. 5 μmol/L mutation detection probe, 0. 25 μmol/L each fluorescence resonance energy transfer （FRET） probe and 1. 5 U FEN1, the background signal of wild-type sample and mutation sample was＆amp;nbsp;dramatically decreased and the background interference to the detecting results was thus eliminated. A total of 21 cases of aldehyde dehydrogenase-2＊2 （ ALDH2＊2 ） , 19 cases of cytochrome p450 2 C19＊2 （ CYP2 C19＊2 ） and 19 cases of CYP2C19＊3 were analyzed
ISSN:0253-3820
DOI:10.11895/j.issn.0253-3820.150051