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目的 扩增位于唐氏综合征关键区域的人肝型磷酸果糖激酶基因 (PFKL基因 ) ,对唐氏综合征进行定量基因诊断。方法 采用定量PCR—微孔板杂交检测PCR产物量的方法 ,对 2 6例唐氏综合征患者及 2 78例正常人外周血DNA标本 ,扩增PFKL基因 ,扩增片段长度为 185bp ;另外 ,将人肌型磷酸果糖激酶基因 (PFKM基因 )作为内参照同时扩增 ,扩增片段长度为 36 5bp ,定量PCR产物用微孔板杂交检测。 结果 2 6例患者 (包括 1例易位型 )PFKM与PFKL扩增产物的OD值比值介于 0 4 0～ 0 6 0之间 ,平均值为 0 5 1;正常人扩增产物的OD值比值介于 0 80～ 1 2 0之间 ,平均值为 1 12 ,两者比较差异显著 (P <0 0 0 1)。对 2 78例正常人进行同样基因扩增和检测无一例假阳性 ,所得结果与染色体核型分析结果完全符合。结论 定量PCR—微孔板杂交检测PFKL基因拷贝数的方法 ,简便、快速、特异 ,可用于唐氏综合征基因诊断
Objective To amplify the human liver phosphofructokinase gene (PFKL) located in key areas of Down Syndrome and to carry out quantitative gene diagnosis of Down Syndrome. Methods The quantity of PCR products was detected by quantitative PCR-microplate hybridization. The PFKL gene was amplified from 26 peripheral blood samples of 26 patients with Down Syndrome and 2 78 normal individuals. The amplified fragment length was 185bp. The human phosphofructokinase gene (PFKM gene) was amplified simultaneously as an internal reference. The amplified fragment was 36 bp in length and the quantitative PCR product was detected by microplate hybridization. Results The OD value of PFKM and PFKL amplification products in 26 patients (including 1 translocation type) ranged from 0 40 to 0 60 with an average of 0 5 1. The OD value of amplification products The odds ratio ranged from 0 80 to 120, with an average of 1 12, showing significant differences (P <0.01). There was no false positive for the same gene amplification and detection in 2 78 normal subjects. The results were in good agreement with the results of chromosome karyotype analysis. Conclusion The method of quantitative PCR-microplate hybridization to detect PFKL gene copy number is simple, rapid and specific and can be used for the gene diagnosis of Down Syndrome