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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 identify fetal ABO genotypes by real-time PCR using fetal DNA (cff DNA) in pregnant women’s plasma. Methods Eighty-nine pregnant women with O-type blood were collected by vein and the free DNA in plasma was extracted. Fetal ABO genotyping was performed using the SYBR real-time PCR method using polymorphisms at positions 261 (nt261) and 796 (nt796). nt261 and nt796 amplified negative plasma DNA after methylation specific enzyme digestion, Taq Man real-time PCR method detection of hypermethylated fetal source RASSF1A gene to determine the presence or absence of cff DNA in plasma DNA. After fetal delivery, serological detection of neonatal ABO blood group, compared with the genetic test results. Results Of the 79 samples, 59 cases were positive for nt261 amplification reaction, of which 33 cases of nt796 primer amplification reaction was positive and 26 cases were negative. 20 cases of nt261 and nt796 amplification negative samples can be detected in the fetus RASSF1A gene to prove the presence of cff DNA. According to the results of serology, the ABO genotypes of 9 fetuses did not match the phenotypes, and the accuracy rate of ABO genotyping in fetuses was 88.6%. The detection accuracy of OO type was 100%, OA type was 76.9% and OB type was 90.9%. Conclusion The detection of fetus ABO genotype by using cff DNA in pregnant women can determine the blood type of fetus during the first trimester and prevent pregnant women with O-fetus from repeatedly reviewing antibody titers and improving the diagnostic accuracy of neonatal hemolytic disease.