荧光定量PCR检测Rh阴性孕妇外周血浆游离DNA中胎儿RhD血型  

Quantitative analysis of fetal RhD genotyping with fetal DNA from RhD-negative maternal plasma

作　　者：王学东[1] 王保龙[1] 叶书来[1] 王兰芳[2] 廖艳秋[1] 沈建军[1] 蒋光明[1] 沈佐君[3] 

机构地区：[1]安徽医科大学附属省立医院输血科,合肥230001 [2]安徽省合肥市妇幼保健院检验科 [3]安徽省临床检验中心

出　　处：《中华检验医学杂志》2008年第10期1147-1152,共6页Chinese Journal of Laboratory Medicine

基　　金：安徽省卫生厅临床医学研究项目资助课题（06B053）

摘　　要：目的探讨荧光定量PCR（fluorescence quantitative polymerase chain reaction，FQ-PCR）技术对Rh阴性孕妇血浆中游离胎儿DNA进行非创性产前诊断胎儿RhD血型的可行性。方法选取78份妊娠11-40周、B超确诊为单胎的Rh阴性孕妇血浆。采用9个短串联重复序列（short tandem repeat，STR）多态性位点及Y染色体性别决定区基因（sex-determining region Y chromosome，SRY）确定胎儿DNA的存在；运用FQ-PCR技术对血浆中游离胎儿DNA进行RHD基因外显子5、7、10和内含子4定量分析，以确定胎儿RhD血型的基因型；其基因型结果与产后新生儿脐血血清学检测结果进行对比分析，回顾性评价胎儿基因定型结果的准确性。结果78份标本中，41份检测到SRY基因，平均浓度为（214．7±120．9）拷贝／ml，产后证实皆为男性。70份FQ-PCR基因定型结果与血清学结果相符，另有5份确定为假阳性，3份基因定型结果不可确定，检测结果总符合率为90％（70／78）。5份假阳性标本通过检测RHD1227A等位基因鉴定了4份RhD放散型，FQ-PCR最终结果准确率达到95％（74／78）。结论应用FQ-PCR方法进行非创性胎儿RhD血型检测可用于新生儿溶血病的预防和诊断。Objective To explore the feasibility of fluorescence quantitative PCR （FQ-PCR） in prenatal diagnosis of the fetal RhD genotyping using free DNA from RhD-negative pregnant women. Methods The fetal RhD gene was amplified from 78 RhD-negative pregnant women with single fetus maternal plasma （ gestation from 11 to 40 weeks）. The existence of fetal DNA was confirmed by amplification of nine different polymorphic short tandem repeat loci （STR） and sex-determining region Y chromosome （SRY） gene. Exon5, 7, 10 and intron 4 were amplified by real-time polymerase chain reaction with TaqMan probe. The results of fetal RhD genotyping were evaluated retrospectively by the serologic analysis of infant cord blood. Results Among the 78 specimens, the SRY positive signals were detected from samples of 41 and were all identified male fetal through sex observation after newborn infants delivered from the women enrolled. The mean concentration of SRY gene reached （214. 7±120. 9） copies/ml. RhD genotyping results of 70 cases were in complete concordance with the results through serological detection of fetal cord blood after delivery. In addition, 5 cases were false-positive. 3 cases were considered inconclusive. The coincidence rate was 90% （ 70/78 ）. From 5 false-positive cases, 4 cases were identified as RhDel phenotype by detecting RHD1227A allele gene. The final accuracy rate of FQ-PCR was 95% （74/78） in the fetal RhD genotyping. Conclusion FQ-PCR analysis for noninvasive prenatal of fetal RhD genotyping could be useful in prevention and diagnosis of hemolytic disease of newborn.

关 键 词：妊娠 Rh—Hr血型系统 产前诊断 聚合酶链反应 

分 类 号：R686[医药卫生—骨科学]