为评价实时荧光PCR法检测 T 细胞受体剪切环(TREC)时,标准曲线法或自动阈值法对不同操作批次重复检测结果稳定性的影响,采用同一批次TREC检测试剂盒重复检测标准质粒中内参基因与靶标TREC的Ct值,同时重复检测一组TREC低值样本的Ct值;分别采用标准曲线法和自动阈值法对检测结果进行分析,比较两种分析方法得到结果(Ct值)的稳定性.对标准质粒和TREC低值样本的检测结果表明,两种分析方法得到的Ct值与两个靶标的标称浓度(log10)均呈线性相关,R2 为 1.00.两种方法获得标准质粒中内参基因Ct平均值,标准曲线法较自动阈值法平均低1.26,标准质粒中TREC基因Ct平均值,前者较后者平均低0.96.对TREC低值样本重复检测结果表明,同一样本标准曲线法获得的两种靶标 Ct 值的变异系数(CV)和极差均低于自动阈值法.表明在以实时荧光PCR法检测TREC靶标从而进行重症联合免疫缺陷(SCID)的筛查时,标准曲线法的分析结果稳定性优于自动阈值法.
Influence of standard curve method and automatic threshold method on the stability of TREC test results
To evaluate the effects of standard curve method or automatic threshold method on the results stability of repeated detection in different batches when real-time fluorescent PCR was used to detect T-cell receptor excision circles(TREC).Ct values of reference gene and target TREC in standard plasmid were repeatedly detected with the same batch of TREC detection kit,and Ct values of a group of samples with low TREC value were repeatedly detected.Standard curve method and automatic threshold method were used to analyze the detection results respectively,and the stability of the results(Ct value)obtained by the two analysis methods was compared.The detection results of standard plasmid and TREC samples with low value show that the Ct values obtained by the two analysis methods were linearly correlated with the nominal concentration(log10)of the two targets,R2 was 1.00.The average Ct values of reference genes in standard plasmid were obtained by the two methods.The average Ct values of TREC gene in standard plasmid using standard curve method were 1.26 lower than those using automatic threshold method,and the average Ct values of TREC gene in standard plasmid using standard curve method were 0.96 lower than those using automatic threshold method.The results of repeated detection of low-value TREC samples show that the coefficient of variation(CV)and range of Ct values of two targets obtained by standard curve method in the same sample were lower than those obtained by automatic threshold method.The findings indicate that the stability of the standard curve method was better than the automatic threshold method when the TREC target was detected by real-time fluorescent PCR for screening severe combined immunodeficiency(SCID).
severe combined immunodeficiency(SCID)T-cell receptor excision circles(TREC)real-time fluorescent PCRstandard curve
NETL
NSTL
万方数据
