应用实时荧光定量PCR检测母血浆胎儿DNA进行RhD基因型产前诊断  

Prenatal determination of fetal RhD genotype by real-time PCR examination of fetal DNA in maternal plasma

作　　者：张逸[1,2] 蒋荔[2] 袁向亮[3] 杨祖菁[1,2] 

机构地区：[1]温州医学院临床医学系,温州325035 [2]上海交通大学医学院附属新华医院妇产科,上海200092 [3]上海交通大学医学院附属新华医院检验科,上海200092

出　　处：《第二军医大学学报》2010年第3期283-287,共5页Academic Journal of Second Military Medical University

基　　金：上海市教委优秀青年教师科研专项基金(jdy06041)~~

摘　　要：目的建立实时荧光定量PCR(real time PCR)技术检测RhD阴性孕妇血浆中游离胎儿DNA进行无创性产前诊断胎儿RhD基因型的方法。方法通过微量DNA抽提技术提取母血浆中胎儿游离DNA,利用Real time PCR检测22例妊娠15～40周的单胎RhD阴性孕妇血浆中胎儿游离DNA,进行男性性别决定基因(SRY)和RhD基因外显子7、10和内含子4的特异性扩增,基因型结果与血清学RhD定型结果对比,评价该技术方法的准确性。结果孕妇血浆中存在游离胎儿DNA。19例RhD真阴性孕妇血浆中,14例均检测到胎儿游离DNA的RhD基因外显子7、10和内含子4的特异性扩增,判断为RhD阳性,基因定型结果与血清学表型相符。3例未检测到胎儿游离DNA的RhD基因特异性扩增,结果与胎儿血清学表型相符。因此检测胎儿游离DNA的RhD基因型准确率达到89.5%。另外2例只检测到RhD基因外显子10扩增,通过新生儿脐血血清学吸收放散试验确定为RhDel型。3例RhDel型孕妇血浆中检测到RhD基因扩增,不适于胎儿RhD基因型的分型。结论实时荧光定量PCR方法检测RhD阴性孕妇血浆中游离胎儿DNA进行无创性诊断胎儿RhD基因型,是一种简便、准确、快速的产前基因诊断的可行性方法,值得临床推广应用。Objective To set up a novel non-invasive prenatal determination technique of fetal RhD genotype by real-time PCR examination of fetal DNA in RhD negative maternal plasma.Methods Plasma fetal DNA was extracted by manual DNA micro-extraction method from the plasma of 22 RhD negative women（15-40 gestation week）.We amplified the exons 7,10 and intron 4 of RhD gene and sex-determining region Y gene（SRY） using real-time polymerase chain reaction.The presence of fetal DNA was confirmed by testing SRY.The results of genotyping and serum RhD status of the newborns were compared to evaluate the accuracy of the present method.Results Cell-free fetal DNA was detected in the maternal plasma samples.Among the 19 RhD negative specimens,14 cases had the specifically amplified exons 7,10 and intron 4 of RhD gene and SRY gene,and the results were confirmed by serological examination of fetal umbilical blood after delivery.Among the 19 specimens,3 cases were not detected the specifically amplified in exons 7,10 and intron 4 of RhD gene and SRY gene,and the results were also confirmed by serological examination of fetal umbilical blood.The accuracy of the present method was 89.5%.SRY detection confirmed fetal DNA presence in maternal plasma in all boys.The other 2 cases only had specifically amplified exons 10 of RhD gene,and the results were confirmed as RhDel phenotype.Real-time PCR easily differentiated pregnant woman whose RBCs had a weak D phenotype（n=3） from truly RhD-negative patients.However,in these cases fetal RhD status cannot be determined.Conclusion Real time PCR detection of fetal DNA in RhD-negative maternal plasma is simple,quick and specific for noninvasive prenatal diagnosis of fetal RhD blood type,which can help to prevent hemolytic disease of newborns.

关 键 词：实时荧光定量PCR 胎儿DNA RhD基因型 产前诊断 外显子 孕妇 可检测 血浆 特异性 血清学 

分 类 号：R714.5[医药卫生—妇产科学]