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目的建立临床检测孕妇外周血中游离胎儿DNA(cell-free fetal DNA,cffDNA)浓度的方法,并比较早孕母血中cffDNA浓度与人绒毛膜促性腺激素(Human chorionic gonadotropin,HCG)及妊娠相关蛋白-A(Pregnancy associated plasma protein-A,PAPP-A)的相关性。方法 (1)用Illumina Hi Seq 2000测序平台进行大规模基因并行测序,利用ELAND软件将其测序得到的原始数据与人类参考基因组进行比对,获得在参考序列上去重复序列的唯一位置的DNA序列,即唯一序列(Unique read),比对后获得的唯一序列不得少于2×106个序列数;(2)运用时间分辨免疫荧光法定量测定PAPP-A含量及游离β-h CG含量。结果建立检测cffDNA浓度的方法,并得出cffDNA,HCG及PAPP-A浓度的相关性;HCG与PAPP-A及cff NDA均呈不显著正相关;PAPPA与cffDNA呈显著正相关。结论成功建立运用第二代测序技术检测孕妇外周血中游离胎儿DNA的方法,该方法将可用于无创性产前诊断,亦可将cffDNA,HCG及PAPP-A三者的联合检测用于早孕期产前筛查。
Objective To establish a method to detect the concentration of free fetal DNA (cffDNA) in peripheral blood of pregnant women and compare the correlation between cffDNA concentration in human maternal blood and human chorionic gonadotropin (HCG) and pregnancy related protein -A (Pregnancy associated plasma protein-A, PAPP-A). Methods (1) Large-scale gene parallel sequencing was performed on the Illumina Hi Seq 2000 sequencing platform. The original data obtained by sequencing were compared with the human reference genome by using ELAND software to obtain the DNA sequence uniquely deduced from the reference sequence. (Unique read). The unique sequence obtained after the comparison should not be less than 2 × 106 sequences. (2) Quantitative determination of PAPP-A content and free β-h CG content by time-resolved immunofluorescence. Results The method of detecting cffDNA concentration was established and the correlations of cffDNA, HCG and PAPP-A concentrations were obtained. There was no significant positive correlation between HCG and PAPP-A and cff NDA, and PAPPA and cffDNA were positively correlated. Conclusion The method of using second-generation sequencing technology to detect fetal DNA in pregnant women’s peripheral blood has been successfully established. The method will be used for noninvasive prenatal diagnosis and combined detection of cffDNA, HCG and PAPP-A in early pregnancy Prenatal screening.