天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (5): 533-536.doi: 10.11958/j.issn.0253-9896.2015.05.023

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连接酶-ELISA反应检测循环DNA基因突变研究

崔海忠 1,肖娜 2,张永平 1,陈大贵 1,唐一通 2△#br# #br#   

  1. 1湖北文理学院医学院枣阳临床学院 （邮编 441200）； 2湖北文理学院医学院分子医学重点实验室
  • 收稿日期:2014-09-10 修回日期:2014-12-18 出版日期:2015-05-15 发布日期:2015-05-25
  • 通讯作者: 唐一通,E-mail： yitongtang@126.com E-mail:yitongtang@126.com
  • 作者简介: 崔海忠 （1972）， 男， 主治医师， 硕士， 主要从事肿瘤学方面研究
  • 基金资助:
    湖北省卫生计生科研基金（WJ2015MB266）； 襄阳市科技局项目（襄科业[2012]43号）； 湖北省教育厅项目（Q20132604）； 湖北省教育厅高校青年教师深入企业计划项目 （XD2014245）

Study for gene mutation detection of circulating DNA with ligase-ELISA reaction

CUI Haizhong1, XIAO Na2, ZHANG Yongping1, CHEN Dagui1, TANG Yitong2△#br# #br#   

  1. 1 Zaoyang Clinical College, Medical College of Hubei University of Arts and Science, Zaoyang 441200, China; 2 Key Laboratory of Molecular Medicine, Medical College of Hubei University of Arts and Science
  • Received:2014-09-10 Revised:2014-12-18 Published:2015-05-15 Online:2015-05-25
  • Contact: TANG Yitong,E-mail： yitongtang@126.com E-mail:yitongtang@126.com

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摘要： 摘要： 目的 研究一种简便、 灵敏的单核苷酸多态性 （SNP） 分型方法， 使其能够在简单实验条件下进行常规的临床样本检测。方法 设计针对突变位点的检测探针， 通过检测探针的连接、 通用扩增、 标记和 ELISA 反应， 根据检测位点对应反应管显色值判定突变位点的基因型。以表皮生长因子受体（EGFR）基因外显子 21 和 18 上的 3 个 SNP 突变位点 L858R、 L861Q 和 G719C 为检测对象， 对 62 例肺癌血浆循环 DNA 样本进行检测， 并与直接测序结果进行比较。结果 通过对 3 个突变位点的检测， 2 种方法均在 L858R 位点检出杂合子突变。直接测序法仅能够明确检出 2 例杂合子突变， 另外 1 例样本因在突变位点出现不明显的套峰而无法明确判定突变类型。而新方法能够明确检出 6 例杂合子突变。结论 建立了一种基于连接酶-ELISA 的简便、 灵敏的 SNP 突变检测方法， 适合于在简单实验条件下对不均一样本进行常规突变检测。

关键词: 单核苷酸多态性, 突变, 基因型, 连接酶, 酶联免疫吸附实验

Abstract: Abstract： Objective To establish a single nucleotide polymorphisms genotyping (SNP) method for a convenient, accu⁃ rate, and routine analysis of clinical samples. Methods Based on the design of oligonucleotide probe, the assay was per⁃ formed through three steps: the conjunction of the detection probe, universal amplification, labeling and ELISA reaction. The genotype of each SNP was revealed by reading signals of each set of reaction tubes. This assay was applied to detect sixtytwo plasma samples of lung cancer for circulating DNA for three SNPs of EGFR, c.2573T>G（L858R） , EGFR, c.2582T>A （L861Q） , EGFR, c.2155 G>T （G719C). Results were compared with those obtained by direct sequencing. Results The het⁃ erozygote mutation was identified for L858R by both methods, although no mutation was detected for L861Q and G719C. Six samples were identified as heterozygotes with the new method, and only two samples were unambiguously identified as het⁃ erozygotes by the direct sequencing. Two additional samples could not be identified as heterozygotes because the peak of mu⁃ tant allele was very low compared with that of wild allele. Conclusion The developed method enabled accurate identifica⁃ tion of SNP in a convenient manner, and which is adapted to routine analysis from heterogeneous samples unambiguously.

Key words: SNP, mutation, genotyping, ligase, ELISA