MGB双荧光探针检测IDH1基因突变

Detection of isocitrate dehydrogenase 1 mutation with MGB dual fluorescent probe

目的建立一种快速检测异柠檬酸脱氢酶1(isocitrate dehydrogenase 1,IDH1)基因单核苷酸多态性的MGB双荧光探针检测方法,并验证该方法检测灵敏度与直接测序方法的一致性。方法构建携带IDH1基因的野生型(R132)和突变型(H132)质粒,并使用质粒优化MGB双荧光探针检测。使用预先已知不同比例的IDH1野生型和突变型质粒作为检测模版,确定IDH1基因突变检测方法的灵敏度。针对56例胶质瘤病人手术切除标本的组织基因组DNA,分别使用直接测序法和MGB双荧光探针检测法,鉴定比较IDH1基因突变类型。结果使用MGB双荧光探针法能够快速准确的检测IDH1基因突变。直接测序检出突变阳性率为26.79%,MGB双荧光探针法检出突变阳性率为30.36%。Kappa检验显示两种方法检测结果一致(P<0.001,K=0.956)。结论 MGB双荧光探针法检测胶质瘤基因组DNA中IDH1基因突变灵敏可靠,操作方便快捷,适合于临床快速分子诊断分析。

Objective To establish a rapid minor groove binder（MGB） dual fluorescent probe method for the detection of the single nucleotide polymorphism（SNP） of isocitrate dehydrogenase 1（ IDH1） gene, and determine the sensitivity of the method and the consistency with the direct sequencing method. Methods The wild-type（R132） and mutant（H132） plasmids were constructed and used to optimize the MGB dual fluorescent probe method. The detection sensitivity of the IDH1 gene mutation was determined using the known proportion of wild-type and mutant plasmids as templates. The genotype DNA of 56 patients with glioma was analyzed by direct sequencing and MGB dual fluorescent probe respectively. The IDH1 gene mutation type was identified and compared. Results IDH1 mutation can be detected by MGB dual fluorescent probe method quickly and accurately. The positivity rate of mutation was 26.79% in direct sequencing and 30.36% in MGB dual fluorescent probe method. Kappa test showed that the results of the two methods were consistent（P〈0.001, K = 0.956）. Conclusions MGB dual fluorescent probe detection can detect the IDH1 gene mutation of glioma genomic DNA sensitively, reliably and rapidly, thus being suitable for clinical rapid molecular diagnostic analysis.