连接酶-ELISA检测K-ras基因突变方法研究

摘要

目的 研究一种简便、灵敏的K-ras基因突变检测方法,使其适合于进行常规突变检测.方法 设计对应检测位点寡核苷酸探针,经探针的连接、扩增、标记及ELISA反应,通过ELISA反应检测值确定突变位点基因型.以K-ras基因12位密码子的G12S、G12R、G12C、G12D、G12A、G12V 6个点突变为检测对象,对72例肺癌血浆循环DNA标本进行检测,并与直接测序结果进行比较.结果 利用建立方法共检出3例标本分别存在G12S、G12R、G12A突变.而通过直接测序未能从标本中检出K-ras突变,表明直接测序方法灵敏度较低,不适合于对循环DNA等不均一标本进行突变检测.结论 建立了一种简便、灵敏的K-ras基因突变检测方法,能够对不均一标本进行常规突变检测.%Objective To research a simple and sensitive K-ras gene mutations detection method in order to be suitable for the routine mutation detection.Methods The corresponding detection locus oligonucleotide probe was designed.By the connection,amplification,labeling and ELISA reaction in probe,the mutation locus genotype was determined by the ELISA reaction detection value.With the six point mutations of G12S,G12R,G12C,G12D,G12A and G12V in 12 codons of K-ras gene as the detection objects,the plasma circulation DNA sample in 72 cases of lung cancer was detected,then the results were compared with those obtained by the direct sequencing.Results Three samples were identified as the G12S,G12R and G12A mutatins by the established method.But no K-ras mutations were detected in the samples by using the direct sequencing,indicating that the direct sequencing had lower sensitivity and was not suitable for the mutation detection of heterogeneous samples such as circulating DNA.Conclusion The simple and sensitive K-ras gene mutation detection method is established and can conduct the routine mutation detection for the heterogeneous samples.