两种实时定量PCR方法检测系统性红斑狼疮患者DNA甲基化状态的比较研究

Comparative study of two kinds of real-time PCR for the detection of the methylation status of DNA

目的:应用两种不同方法检测系统性红斑狼疮(SLE)患者CD4^+T细胞p16基因甲基化状态,比较两种检测方法的差异。方法:采用以Taqman探针为基础的MSP法(方法1)和以SYBR Green Ⅰ为基础的MSP法(方法2)分别检测40例SLE患者和20例正常人CD4^+T细胞中p16基因启动子区甲基化状态。结果:Taqman探针方法的结果为:SLE患者CD4^+T细胞p16基因甲基化阳性率(35.7%,10/28)高于对照组(10%,2/20),两者比较差异有统计学意义(x^2=4.11,P<0.05)。SYBR Green Ⅰ方法的结果为:患者组和对照组的CD4^+T细胞的p16基因均呈高甲基化状态,应用t检验分析发现P>0.05,二者无统计学差异。结论:Taqman探针法消除了引物二聚体和非特异性扩增对试验结果的影响,提高了结果的特异性和准确性,被证明是进行DNA甲基化状态检测的可靠方法。

Objective： To use two kinds of real-time PCR to detect the methylation status of p16 gene in CD4^＋ T cell derived from SLE patients and compare the effect of the two methods. Methods：P16 promotor methylation in CD4^＋ T cell was detected with both the Taqman probe based real-time PCR technology and the SYBR Green Ⅰ based real-time PCR technology（method 2） in 40 SLE patients and 20 healthy controls. Results：The result of Taqman probe method showed that the rate of p16 gene hypermethylation was higher in SLE patients（35.7%） than in that of the controls （10%）（Х^2=4.11, P=0.04〈0.05）. The result of SYBR Green method showed that there was no significant difference between the patients and controls （P〉0.05）. Conclusions：Taqman probe method can effectively eliminate the effect of the primer-dimers （PDs） and nonspecific amplieation on the process of PCR, and increase the specificity and accuracy of the result. This method can be used to detect the the methylation status of DNA.