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目的建立三重TaqMan实时荧光定量PCR体系,快速诊断21、X染色体数目异常。方法选取21号染色体唐氏综合征特异区域(DSCR3)和X染色体1113KDNA序列范围分别作为21号和X染色体目的基因,同时选择12号染色体上磷酸甘油醛脱氢酶基因(GAPDH)为参比基因,分别设计引物和TaqMan探针,经PCR反应体系优化,采用单管三重及2-ΔΔCt相对定量数据分析,根据基因拷贝数变化诊断21号和X染色体数目异常,并进行方法学应用评价。结果通过优化,模板量在5～30ng/μl时3个基因都可以得到有效扩增,目的基因和参照基因扩增效率基本一致,均接近100%。采用上述体系检测24例正常标本和20例异常标本(包括血液和羊水标本),正常标本的21号2-ΔΔCt值均为2,而唐氏综合征患者均为3;正常男性的X染色体2-ΔΔCt值均为1,而正常女性均为2。其临床标本检测结果的敏感性、特异性均达100%。结论建立的三重TaqMan实时荧光PCR相对定量法快速诊断21和X染色体数目异常方法 ,稳定性、可靠性和实用性均较好。
Objective To establish a triple TaqMan real-time fluorescence quantitative PCR system for rapid diagnosis of 21 and X chromosome abnormalities. Methods DSCR3 and 1113K DNA sequence of chromosome 21 were selected as the target genes of chromosome 21 and X. At the same time, the glycoaldehyde-6-phosphate dehydrogenase gene (GAPDH) on chromosome 12 was selected as the reference gene , Primers and TaqMan probes were designed and optimized by PCR reaction system. The single-tube triple and 2-ΔΔCt relative quantitative data analysis were used to diagnose 21 and X chromosome abnormalities based on gene copy number changes, and the methodological application was evaluated. The results showed that all the three genes could be effectively amplified when the amount of template was between 5 ~ 30ng / μl. The amplification efficiency of target gene and reference gene were almost the same, both of which were close to 100%. Twenty-four normal specimens and 20 specimens of abnormalities (including blood and amniotic fluid specimens) were detected by the above-mentioned system. The 2-ΔΔCt values of No. 21 were all 2 in normal specimens and 3 in Down’s syndrome specimens. The normal male X chromosome 2 -ΔΔCt values were 1, while normal women were 2. The sensitivity of the clinical specimen test results, the specificity of up to 100%. Conclusion The established triple TaqMan real-time fluorescent PCR relative quantification method for rapid diagnosis of 21 and X chromosome number anomalies, stability, reliability and practicality are good.