一种检测多种NPM1突变体的ARMS-PCR方法的建立

本研究旨在建立一种简单、灵敏的,能检测多种NPM1突变体的方法,以降低NPM1突变的漏检率。通过构建NPM1基因野生型和最常见的突变型A、B、C、D重组质粒作为检测对象,根据NPM1不同突变体碱基排列方式不同,设计1对特异性引物,使得上游引物3’末端的碱基与突变体A、B、C、D匹配,而与野生型NPM1不匹配;通过条件优化,建立检测多种NPM1突变的ARMS-PCR的方法。通过对该方法的检测范围、灵敏度的评估及与直接测序法的比较,判断该方法的可行性。结果表明,成功构建NPM1基因野生型和突变型A、B、C、D 5种重组质粒,经测序鉴定和目的片段一致。ARMS-PCR方法可以检测到ABCD 4种突变体,检测范围在103copies/ml-109copies/ml,其灵敏度为0.01%,而当突变体含量低于10%时采用直接测序则检测不出突变。结论:本研究建立了一种可以检测NPM1 4种高频突变的ARMS-PCR方法。该方法灵敏度高,可以检出95%以上的突变,为临床检测NPM1基因突变提供一种新型的检测方法。

ARMS-PCR Method for Detecting Multiple NPM1 Mutations

This study was aimed to establish a simple,sensitive detection method for multiple NPM1 mutations,so as to reduce the omission ratio of NMP1 mutant detection.Recombinant plasmids containing wide-type NPM1 and the most common mutations（A,B,C,D） were constructed as the detection objects.The ARMS-PCR for detecting multiple NPM1 mutations was established through designing a pair of specific primers whose 3′ end base matched with four mutants（A,B,C,D）,but did not mached with wild type NPM1 according to the different base sequence of NPM1 mutants.The feasibility of the ARMS-PCR method was evaluated by assessing the detection range and the sensitivity and comparing with direct sequencing.The results showed that the recombinant plasmids were constructed successfully by restriction analysis and DNA sequencing.The four mutants but not wild type NPM1 were detected by using ARMS-PCR,the detection range of the method was 103copies/ml-109copies/ml and the sensitivity was 0.01%,while the direct sequencing method could not detect the mutations if mutation was less than 10%.It is concluded that the high sensitive ARMS-PCR is established for detecting the four mutations of NPM1 and more than 95% mutants can be detected by this method,providing a new detection method for clinical NPM1 gene mutant.