摘要
目的采用实时定量PCR(real-time PCR)通过孕妇血浆中的胎儿游离DNA(cff DNA)鉴定胎儿ABO血型基因型。方法静脉采集79名孕妇O型血液,提取血浆中的游离DNA。采用SYBR real-time PCR方法,利用261位点(nt261)和796位点(nt796)的多态性进行胎儿ABO基因分型。nt261和nt796扩增反应阴性的血浆DNA经甲基化特异性酶消化后,用Taq Man real-time PCR方法检测高甲基化胎源RASSF1A基因,来确定血浆DNA中是否存在cff DNA。待胎儿分娩后,血清学检测新生儿ABO血型,与基因检测结果比对。结果 79例标本中59例nt261扩增反应为阳性,其中33例nt796引物扩增反应为阳性,26例为阴性。20例nt261和nt796扩增反应阴性的标本中均可检测到胎儿RASSF1A基因,证明存在cff DNA。参照血清学结果,9例胎儿ABO基因型与表型不符,胎儿ABO基因分型准确率为88.6%,其中OO型检测准确率为100%,OA型为76.9%,OB型为90.9%。结论利用孕妇血浆中cff DNA检测胎儿ABO基因型可以在孕早期确定胎儿血型,避免怀有O型胎儿的孕妇多次复查抗体效价,并提高新生儿溶血病诊断的准确性。
Objective To establish a protocol to detect fetal ABO genotypes using the cell-free fetal DNA （cffDNA） in maternal plasma by real-time PCR. Methods The cell-free DNA was extracted from the plasma of 79 blood group O preg- nant women. Fetal ABO genotyping w~ performed by SYBR real-time PCR with the allele-specific primers at nucleotide posi- tion （nt） 261 of exon 6 and nt796 of exon 7. The samples with negative amplification of nt261 and nt796 were treated by methylation sensitive restriction enzyme digestion. Hypermethylated RASSF1A gene of fetal origin was detected by TaqMan re- M-time PCR to ascertain existence of cffDNA. The fetal ABO genotyping results were compared with the serological ABO blood groups of the newborns. Results In the total of 79 plasma samples, 59 samples showed positive amplification at nt261, in which 33 samples showed positive amplification and 26 samples showed negative amplification at nt796. The amplification signal of fetal RASSF1A gene was detectable in the 20 samples with no amplification at nt261 and nt796, which confirmed the existence of cffDNA in these samples. According to the serological ABO blood groups of the newborns, there were nine false-diagnosed cases. The diagnostic accuracy of fetal ABO gnnotyping was 88. 6%, in which the accuracy of fetal genotype 00, OA and OBwas 100%, 76.9% and 90. 9%, re- spectively. Conclusion Our protocol could determine fetal ABO gcnotypes in early pregnancy, avoiding the follow-up anti- body level tests in pregnant women carrying blood group 0 fetuses and facilitating the diagnosis of hemolytic disease of the fe- tus and newborn （HDFN）.
出处
《中国输血杂志》
CAS
北大核心
2016年第2期127-130,共4页
Chinese Journal of Blood Transfusion
