应用孕妇外周血cff-DNA检测胎儿RHD血型基因的方法学研究  

Methodological Study on the Application of Maternal Peripheral Blood cff-DNA for the Detection of Fetal RHD Blood Group Gene

作　　者：任道菊 杨金华 李小薇 肖军 李翠莹 REN Daoju;YANG Jinhua;LI Xiaowei;XIAO Jun;LI Cuiying(Department of Blood Transfusion,Air Force Medical Center,Air Force Medical University,Beijing 100142;2Graduate School of PLA Medical College,Beijing 100853)

机构地区：[1]空军军医大学空军特色医学中心输血科,北京100142 [2]解放军总医院医学院研究生院,北京100853

出　　处：《临床输血与检验》2025年第2期163-169,共7页Journal of Clinical Transfusion and Laboratory Medicine

基　　金：空军特色医学中心青年人才项目(No.2022YXQN028);空军军医大学临床研究计划项目(No.2024LC2406)资助。

摘　　要：目的研究一种新型应用孕妇外周血胎儿游离DNA(cell free fetal DNA,cff-DNA)检测胎儿RHD血型基因的无创检测方法,并分析该方法的可行性及准确性。方法以2023年8月—2024年8月本医学中心建档的RhD阴性孕妇为研究对象,采集孕妇5~8 mL外周血,首先对母亲RHD基因型进行检测,采用吸附柱法提取孕妇外周血中的基因组DNA,用PCR-SSP法检测母亲RHD基因分型,筛选出符合纳入标准的孕妇;然后采用磁珠法提取孕妇血浆中的cff-DNA,对提取得到的cff-DNA先进行质控检测,使用甲基化敏感限制性内切酶处理cff-DNA,酶切后扩增RASSF1A基因及β-actin基因,以确保提取到胎儿来源的cff-DNA;再进行胎儿RHD血型基因检测,运用巢式结合荧光探针的qPCR方法,扩增RHD第4、7、10外显子,确定胎儿RhD血型;最后对胎儿cff-DNA RHD血型基因检测结果准确性进行验证,追踪出生后新生儿血型,以其为金标准,判断孕妇外周血cff-DNA RHD血型基因检测结果的准确性。结果收集到4例RhD阴性孕妇样本,进行母亲RHD基因分型,结果3例为RHD全缺失,1例为RHD-CE(2-9),均符合纳入标准;对孕妇外周血中提取得到的cff-DNA,酶切后质控验证,RASSF1A基因均为阳性,β-actin基因均为阴性,均提取到cff-DNA;运用cff-DNA检测胎儿RHD血型基因,结果显示4例RHD第4、7、10外显子均为阳性,判断胎儿RhD血型为RhD阳性;追溯4例新生儿,新生儿血型血清学结果为RhD阳性。结论这种新的孕妇外周血cff-DNA检测胎儿RHD血型基因的方法可行,检测结果与出生后的新生儿血型结果一致,其对早期诊断RhD相关HDFN及指导RhD阴性孕妇应用抗D免疫球蛋白具有重要意义。Objective This study is to establish a novel non-invasive testing method to detect fetal RHD blood group genes of cell free fetal DNA(cff-DNA)from peripheral blood of pregnant women,and to analyze the feasibility and accuracy of the method.Methods RhD-negative pregnant women who were enrolled in our medical center from August 2023 to August 2024 were used as the study subjects,and 5~8 mL of peripheral blood was collected from pregnant women,firstly,the mother's RHD genotype was detected,and the genomic DNA in the peripheral blood of the pregnant women was extracted by the adsorption column method,and the genomic DNA in the peripheral blood of the pregnant women was extracted by the sequence specific primer polymerase chain reaction(Polymerase Chain Reaction).Reaction-Sequence Specific Primer(PCR-SSP)method was used to detect the RHD genotypes of mothers,and pregnant women who met the inclusion criteria were screened out;then cff-DNA in the plasma of pregnant women was extracted by the magnetic bead method,and the extracted cff-DNA was first subjected to quality control testing,and cff-DNA was processed using methylation-sensitive restriction endonuclease,and RHD genotypes were amplified after enzymatic digestion.DNA,methylation-sensitive restriction endonuclease was used to treat cff-DNA,and the RASSF1A andβ-actin genes were amplified after digestion to ensure that cff-DNA of fetal origin was extracted;then the fetal RHD blood group gene test was performed,and the nested Taqman probe was used in the realtime quantitative polymerase chain reaction(qPCR)to detect the cff-DNA.The qPCR method was used to amplify exons 4,7 and 10 of RHD to determine the fetal RhD blood group;finally,the accuracy of the fetal cff-DNA RHD blood group gene test was verified,and the newborns'blood group after birth was tracked,which was used as a gold standard control to determine the accuracy of the maternal peripheral blood cff-DNA RHD blood group gene test.Results The samples from four RhD-negative pregnant women were collected for mo

关 键 词：cff-DNA 胎儿RHD血型基因 RHD阴性 胎儿新生儿溶血病 巢式PCR 

分 类 号：R714.5[医药卫生—妇产科学] Q789[医药卫生—临床医学]