摘要
目的建立检测人外周血单个核细胞(PBMC)中FOXP3mRNA的方法。方法跨内含子设计外引物,并根据扩增的片段,设计一对内引物和TaqM an探针,采用套式实时荧光定量PCR(nested-real-tim e-PCR)方法。首次PCR扩增用减少引物浓度及循环次数的方法以增加其模板的特异性,再行第二次PCR扩增作实时荧光定量检测,用FOXP3和β-actin标准品作标准曲线,以二者比值作为FOXP3mRNA表达水平指标。结果重复6次PCR,6次试验C t的总平均值为14.66,平均变异系数为5.81%。C t值和模板起始浓度对数值之间具有较好的线性关系(R2=0.999);检测最低浓度可达100拷贝。结论nested-re-al-tim e-PCR检测PBMC中FOXP3mRNA的方法简单且重复性好,高度敏感且特异,可作为评价免疫功能及某些疾病治疗效果观察的临床常用方法。
Objective To develop a nested-real-time PCR method for the detection of FOXP3 mRNA in human peripheral blood mononuclear cells （PBMC）. Methods The outer primers were designed by span intron, and the inner primers and TaqMan probe were designed according to the amplified sequence. During the nested real-time PCR, decrease primers concentration and cycle times were decreased in first PCR amplification to increase specific templates ,then quantitative real-time fluorescence was detected in the second PCR amplification. The standard curve was created by FOXP3 and beta-actin recombined plasmid DNA respectively, and the results were presented as the ratios of FOXP3 mRNA to beta-actin mRNA. Results Total mean Ct（threshold cycle） in 6 tests was 14.66. The mean CV（ coefficient of variation） was 5.81%. The standard curve showed a fine linear relationship between Ct and template concentration, and the correlation coefficient was 0.999. The sensitivity was 100 times higher than normal real-time PCR by reach 100 copy. Conclusions Nested-real-time-PCR for the detection of FOXP3 mRNA in human PBMC is a simple, repeatable, high specific and sensitive method to evaluate immune function and observe therapeutic effects in certain diseases for clinical routine application.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2006年第4期289-291,共3页
Chinese Journal of Clinical Laboratory Science
