孕母血中cffDNA浓度与HCG及PAPP-A相关性的研究

Study on the correlation of cffDNA concentration,HCG and PAPP-A in maternal blood

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 for detecting the concentration of cell-free fetal DNA（cffDNA）in peripheral blood of pregnant women,and to compare the correlation of cffDNA concentration,HCG among PAPP-A in maternal blood during the first trimester of pregnancy. Methods：（1）To carry out the large-scale parallel sequencing with Illumina Hi Seq 2000 sequencing platform,to compare the original data by sequencing and the human reference genome using ELAND software,the DNA sequence of the unique sequence（Unique read）,which is the reference sequence for removal of repetitive sequences,has been obtained. The Unique read is not less than 2×10^6 sequences;（2）To determine the content of PAPP-A and Free betah CG using the method of time resolved fluorescence immunoassay. Results：the method of detecting cffDNA concentration has been successfully established,and the correlation of cffDNA,HCG and PAPP-A concentration has been obtained;HCG and PAPP-A,HCG and cff NDA were no significantly positive correlation,PAPPA and cffDNA was significantly positive correlation. Conclusion：The method of detecting fetal DNA in peripheral blood of pregnant women has been successfully established using second generation sequencing,the method can be used for noninvasive prenatal diagnosis,can also be cffDNA,the combined detection of cffDNA,HCG and PAPP-A are also used for early prenatal screening.