扩增人肝型磷酸果糖激酶基因对唐氏综合征进行定量基因诊断的研究

Amplification PFKL gene in the Down's syndrome critical region for quantitative genetic diagnosis

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 。

Objective To disscuse the value of amplificated PFKL gene in the Down syndrome critical region for quantitative genetic diagnosis.Methods PFKL gene was amplified in the peripheral blood DNA samples of 26 patients with Down's syndrome and 278 normal individuals by quantitative polymerase chain reaction.A 185bp specific fragment was obtained.Meanwhile,PFKM gene (365bp) was also amplified as an inner control.The PCR products was measured by enzyme linked oligonucleatide assay (ELOSA).Results The optical density ratio of PCR products of PFKM gene and PFKL gene in patients ranged from 0 40 to 0 60 (mean 0 51).While in normal people,it ranged from 0 80 to 1 20 (mean 1 12).There was significant difference between patients and normal individuals in the optical density ratio (P<0 001).No one case of false positive result was found in normal 278 cases.The results were consistent with the chromosome karyotype analysis.Conclusion Detection of PFKL gene copies by quantitative PCR is an easy,quick,efficient method for genetic diagnosis of Down's syndrome.