建立一种N-ras突变检测的“凸背”引物荧光PCR新技术

Establishment of a new "convex dorsal" primer fluorescence PCR method to detect the mutation of N-ras

目的建立一种简便、省时、有效的N-ras突变检测的＂凸背＂引物荧光PCR新技术。方法对比＂凸背＂引物荧光PCR新技术和Sanger测序法：通过对混有不同比例的N-ras基因热突变G13D（GGT〉GAT）质粒的样本进行对照检测来比较两者的灵敏度;对97例急性髓细胞白血病（AML）患者的外周血的DNA进行N-ras的G13D突变检测,统计两者的符合率。结果 ＂凸背＂引物荧光PCR新技术能检测出混有5%突变质粒型的样本,其灵敏度达5%,高于Sanger测序法的10%~20%。在97例AML患者的外周血的DNA中,＂凸背＂引物荧光PCR新方法和Sanger测序法检测到N-ras基因G13D突变分别为19例和18例,符合率达94.7%。结论 ＂凸背＂引物荧光PCR新技术比测序法更为快速、简单,有更高的灵敏度,容易实现。

Objective To establish a simple, rapid and effective ＂convex dorsal＂ primer fluorescence PCR to detect the mutation of N-ras. Methods The N-ras mutation of G13D （GGT 〉 GAT） was detected by ＂convex dorsal＂ primers fluorescent PCR method and Sanger sequencing method, respectively. The samples which were mixed with different proportions of the N-ras mutant plasmid（two methods） were detected, and their coincidence rates were calculated. 97 acute myeloid leukemia （AML） peripheral blood samples were tested, and their coincidence rate was calculated. Results The samples which were mixed with 5% N-ras mutant plasmid can be detected by the ＂convex dorsal＂ primers fluorescent PCR method. Its sensitivity （5%） was higher than Sanger sequencing method＇s. In 97 acute myeloid leukemia（AML） peripheral blood samples, 19 case with N-ras mutations（G13D） were detected by ＂convex dorsal＂ primers fluorescent PCR method, and 18 case with N-ras mutations（G13D） were detected by Sanger sequencing method. The coincidence rate is 94.7%. Conclusion The ＂convex dorsal＂ primers fluorescent PCR method is more convenient, high sensitivity than sequencing method, and easily achieved.