孕妇血浆中游离胎儿DNA在产前诊断中的初步应用

目的探讨孕妇血浆中游离胎儿DNA在非创伤性产前诊断中的可行性。方法用柱分离法提取46例孕妇血浆中胎儿DNA,用巢式聚合酶链反应(PCR)技术扩增其胎儿SRY基因,并引入内参照基因ATL1特异序列。结果 28例孕男胎的孕妇血浆中有26例经巢式PCR扩增出SRY基因,18例孕女胎的孕妇血浆中有17例经巢式PCR只扩增出ATL1基因。灵敏度和特异度分别为92.9%(26/28)和94.4%(17/18),总符合率为93.5%(43/46)。结论应用孕妇血浆中胎儿DNA作产前诊断灵敏度和特异度较高,是一种非创伤性产前诊断方法,具有广泛的临床应用前景。     

孕妇血浆中胎儿游离DNA用于产前诊断的研究进展

产前诊断是指在胎儿出生前利用各种方法诊断胎儿是否患有某种遗传病或先天性疾病的一种手段,是出生缺陷工作的重要任务,对于提高我国出生人口的素质有着特别重要的意义。现在的产前诊断技术大部分都属侵入性检查,有引起孕妇     

实时荧光定量PCR检测早产孕妇血浆中游离胎儿DNA

近年来研究学者证实孕妇血浆中存在着胎儿DNA,这一发现有极重要的潜在临床应用价值,这为非损伤性的疾病诊断及孕期监测提供了一种新的工具。本研究应用实时荧光定量聚合酶链反应（RQ-PCR）检测出现早产宫缩的孕妇血浆中游离胎儿DNA来确定胎儿DNA在母血浆中的存在,以期为孕期监测和早产的病理生理过程的进一步研究奠定基础。     

实时荧光定量PCR检测早产孕妇血浆中游离胎儿DNA

近年来研究学者证实孕妇血浆中存在着胎儿DNA[1],这一发现有极重要的潜在临床应用价值,这为非损伤性的疾病诊断及孕期监测提供了一种新的工具。本研究应用实时荧光定量聚合酶链反应(RQ-PCR)检测出现早产宫缩的孕妇血浆中游离胎儿DNA来确定胎儿DNA在母血浆中的存在,以期为孕期监测和早产的病理生理过程的进一步研究奠定基础。一、材料和方法1.对象本院2004年1月~2005年2月收治的自发性规则宫缩的孕妇102例,其中初次妊娠者69例,2次妊娠者31例,多次妊娠者2例。孕妇妊娠孕周为28~37周。孕妇年龄2 l~35岁。取2名健康男性及2名未孕且无妊娠…     

应用套式聚合酶链反应检测孕妇外周血中胎儿DNA

确定孕妇外周血中是否存在胎儿细胞以及不同孕期对他的影响。采用套式聚事酶链反应技术，用人Ｙ染色体ＤＮＡ特异基因（ｓｅｘｄｅｔｅｒｍｉｎｉｎｇｒｅｇｉｏｎＹ，ＳＲＹ）作为研究孕妇外周血中胎儿ＤＮＡ的一个基因标本，结果在妊娠早期（８周＾＋３）开始到妊娠晚期（３８周）的孕妇血循环中均能发现源于胎儿的有核细胞，男性胎儿阳性结果的预测值在早、中、晚三个不同孕期分别为３／５，６／８，５／６，试验敏感度为：可在相     

检测孕妇血浆中游离胎儿DNA行产前诊断的研究进展

及早获取胎儿组织进行产前诊断,对于有效预防和控制新生儿遗传性疾病的发病率及死亡率十分重要。传统的产前获取胎儿组织的方法,如羊膜腔穿刺和绒毛膜取样等均为创伤性检查,对胎儿和孕妇都有一定的危险。1997年,Lo等首次证实孕妇血浆(清)中存在游离的胎儿DNA(fetal DNA,fDNA),并将其用于性连锁遗传病的产前诊断,为无创性产前诊断开辟了新的途径。本文就近年来利用血浆fDNA行产前诊断的进展及存在的问题作一简要综述。1孕妇血浆fDNA的基本特性孕妇血浆中fDNA的基本特性为:①含量高于孕妇血液中胎儿细胞的DNA。孕妇血浆总的游离DNA是fDNA和母体自身DNA的混合物。妊娠早期fDNA的平均含量为25．4     

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

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.     

孕妇血浆游离胎儿 DNA 检测在无创产前诊断中的应用价值研究

目的：探讨孕妇血浆游离胎儿 DNA 检测在无创产前诊断中的价值。方法2011年1月至2013年12月该院46例孕妇作为研究对象,通过柱分离法获得孕妇的血浆 DNA ,并通过聚合酶链反应（PCR）技术对孕妇外周血浆中游离胎儿 DNA 的性别决定基因（SRY）进行检测,并通过 ATL1基因鉴别胎儿的性别。结果46例孕妇中28例产男性婴儿,18例产女性婴儿,其中男性胎儿中的26例能通过 PCR 技术扩增出 SRY 基因,而女性胎儿中有17例经过 PCR 技术扩增出 ATL1基因,其特异度为94．4％（17／18）,而灵敏度为92．9％（26／28）,检测的总符合率为93．5％（43／46）。结论将孕妇血浆游离胎儿 DNA 检测应用到产前检查检测中,其灵敏度和特异度都较高,且检查过程中无创伤,能减少不良事件的发生概率,值得临床推广和应用。     

孕妇血浆中的游离胎儿核酸

目前 ,产前诊断主要是利用胎儿的组织来检测一些遗传性疾病 ,这对于预防及控制新生儿发病率及死亡率具有十分重要的临床意义。然而 ,损伤性的诊断如羊水和绒毛膜穿刺等方法都有机会对胎儿和孕妇造成一定的危害[1] 。因此 ,这些方法通常仅用于那些怀疑怀有遗传性疾病胎儿的高风险孕妇。鉴于损伤性产前诊断的局限性 ,故非损伤产前性诊断的方法 ,具有极大的发展空间和潜力。其中 ,母亲与胎儿之间存在细胞相互渗透的现象已为研究所证实[2 3] ,而孕妇血液中的胎儿细胞为有效地进行非损伤性诊断 ,提供了有价值的遗传物质[4 ] 。然而孕妇血液中的…     

利用孕妇血浆中胎儿游离DNA进行无创伤性产前基因诊断的研究进展

孕妇血浆中胎儿游离DNA,源于胎儿细胞和/或胎盘的凋亡,经核酸内切酶选择性地剪切为313 bp以下的短片段分子,因相对含量丰富,成为了当前无创伤性产前分子遗传诊断中胎儿DNA的重要来源,业已开展了胎儿性别鉴定、RhD阴性孕妇的Rh（D）基因检测、胎儿非整倍体病的诊断、STR遗传标记检测等应用研究。根据胎儿游离DNA的浓度、纯度、分子片段大小分布的特征和检测的靶基因,采用相应的分子基因诊断策略和实验设计,限制扩增产物的片段长度,有助于提高无创伤性产前胎儿分子遗传诊断的成功率。当前,大规模并行基因组测序技术为直接利用胎儿游离DNA进行无创伤性产前基因诊断开辟了新途径。     

Illumina测序技术在母血检测胎儿非整倍体中应用

目的研究Illumina测序技术在母血中检测胎儿非整倍体的可行性,及其在无创性产前诊断中的应用前景。方法 68例孕12-39周具有产前诊断指征的单胎妊娠孕妇抽取外周血进行离心、提取血浆中游离DNA,运用Illumina Hiseq2000测序技术进行DNA测序及统计分析。结果 68例孕妇通过Illumina测序技术检测出3例21三体,2例18三体、1例13三体及1例47,XYY。因为濒死胎儿羊水脱落细胞活力极低而致羊水培养失败,传统型染色体核型分析未检测出1例21三体。结论 Illumina测序作为一种无创性产前诊断技术,可准确地筛查21、18、13、X、Y等染色体非整倍体异常,具有安全、准确率高及假阳性率低的优点,值得临床推广。     

孕周对产前超声诊断胎儿腹腔积液及胎儿结局的影响

目的 观察孕周对产前超声诊断胎儿腹腔积液及胎儿结局的影响。方法 根据首诊胎儿腹腔积液时孕周将58例超声诊断胎儿腹腔积液孕妇（58胎）分为2组，Ⅰ组（<24周）20例，Ⅱ组（≥ 24周）38例；对比2组产前诊断结果及胎儿结局。结果 58例中，54例（54/58，93.10%）产前诊断明确，4例为胎儿特发性腹腔积液（单纯性腹腔积液，未合并其他异常）。Ⅰ组产前诊断结果主要为胎儿消化系统疾病及水肿，Ⅱ组主要为胎儿消化系统疾病、水肿、泌尿生殖系统异常及循环系统异常等；Ⅰ组与Ⅱ组产前诊断差异无统计学意义（P均>0.05）。Ⅰ组12例（12/20，60.00%）终止妊娠，7例（7/20，35.00%）胎儿结局好；Ⅱ组10例（10/38，26.32%）终止妊娠，5例新生儿出生后7天内死亡（5/38，13.16%），20例（20/38，52.63%）胎儿结局好。Ⅰ组终止妊娠率高于Ⅱ组（χ²=6.32，P=0.01）。结论 孕周对产前诊断腹腔积液及胎儿结局均存在影响。     

产前超声评估胎儿胰腺

Objective The aim of the study was to assess the sonographic feasibility of measuring the fetal pancreas and its normal development throughout pregnancy. Methods Images of fetal pancreas of single-fetal non-diabetic pregnant women between 14 and 38 weeks of gestation were collected and followed up.The relationship between fetal PC and GA and AC was observed. Results 425 fetal pancreas were recruited in this study.The overall satisfactory rate was 62.35% (265/425),the circumference of 265 fetal pancreas was measured. Through correlation analysis, the PC was positively correlated with GA and AC (R2=0.67, 0.65 P<0.01),respectively. The normal reference range of PC of 18 to 34 weeks of gestation was given.During this study,one case of the AP was found. Conclusion In this study,we assessed the feasibility of sonography for measuring the fetal pancreas and established a normal reference range for the fetal PC throughout pregnancy.     

Noninvasive determination of fetal RHD status by examination of cell-free DNA in maternal plasma

BACKGROUND: Cell-free fetal DNA in maternal plasma opens the way for routine risk-free diagnosis of fetal D status of D- mothers. The focus was on accuracy of RHD typing and confirmation of fetal DNA in maternal plasma while RHD was not detected. STUDY DESIGN AND METHODS: Plasma DNA was extracted (by manual and/or automatic method) from 255 D- pregnant women and amplified in exons 7 and 10 and intron 4 of RHD gene with real-time polymerase chain reaction. The presence of fetal DNA was confirmed by testing SRY and, when negative, by one of 11 different polymorphisms found in the father but not in the mother. The results were compared with the D status of the newborns. RESULTS: After exclusion of 25 cases (10%) because of material shortage, in 230 cases (90%) available for complete study, the predictive value of the procedure of fetal RHD testing (RHD genotyping plus confirmation of fetal DNA) was 99.6 percent. SRY detection confirmed fetal DNA presence in maternal plasma in all boys, whereas the detection of various polymorphisms in all girls but one. CONCLUSIONS: Fetal RHD genotyping from maternal plasma may be used with confidence, although additional polymorphisms for confirmation of fetal DNA should be included for 100 percent predictive value (instead of 99.6%).     

Evaluation of prenatal RHD typing strategies on cell-free fetal DNA from maternal plasma

BACKGROUND: The discovery of cell-free fetal DNA in maternal plasma led to the development of assays to predict the fetal D status with RHD-specific sequences. Few assays are designed in such a way that the fetus can be typed in RHDpsi mothers and that RHDpsi fetuses are correctly typed. Owing to the limited knowledge about the mechanism responsible for the presence of fetal DNA in maternal plasma, precautions in developing prenatal genotyping strategies must be made. STUDY DESIGN AND METHODS: Real-time quantitative (RQ)-polymerase chain reaction (PCR) assays were developed for prenatal diagnostic use with cell-free fetal DNA from maternal plasma. An RQ-PCR assay on RHD exon 5 (amplicon 361 bp), negative on RHDpsi, was developed with genomic DNA and evaluated with cell-free fetal DNA. A previously published RHD exon 5 RQ-PCR (amplicon 82 bp) was duplexed with an in-house developed RHD exon 7 RQ-PCR and evaluated with cell-free fetal DNA from pregnant D-RHDpsi+ women. RESULTS: The RHD exon 5 361 bp assay showed on cell-free plasma DNA from D- women carrying a D+ fetus, low amplification levels, resulting in high Ct values and false-negative results. Owing to fragmentation of cell-free plasma DNA, too few DNA stretches of sufficient length (> 360 bp) are present. The RHD exon 5 82 bp and exon 7 RQ-PCR duplex was evaluated with RHDpsi+ cell-free plasma DNA and showed complete specificity and maximal sensitivity. CONCLUSION: Assays designed for prenatal genotyping should be developed and evaluated on cell-free plasma DNA. Prenatal RHD typing is accurate with the RHD exon 5 82 bp and exon 7 duplex strategy.     

Application of fetal DNA in maternal plasma for noninvasive prenatal diagnosis

Prenatal diagnosis of fetal genetic conditions is a standard part of modern obstetric care. Many of the current methods rely on invasive methods and are associated with an inherent risk of fetal loss. Consequently, there has been a long-term goal for development of noninvasive prenatal diagnostic methods. In 1997, the presence of fetal DNA in maternal plasma was first discovered through the detection of Y-chromosome-specific sequences in the plasma of women conceived with male fetuses. This discovery has opened up new possibilities in the development of noninvasive prenatal diagnostic methods through a source of fetal genetic material that could be conveniently accessible simply through the collection of a maternal peripheral blood sample. To date, there have been numerous reported applications, including fetal RhD genotyping, prenatal diagnosis of sex-linked disorders, paternally inherited genetic diseases and some pregnancy-associated conditions, including preeclampsia. More recently, there have been significant new developments with expanding number of potential applications.     

Non-invasive prenatal diagnosis of fetal aneuploidies using massively parallel sequencing-by-ligation and evidence that cell-free fetal DNA in the maternal plasma originates from cytotrophoblastic cells

Blood plasma of pregnant women contains circulating cell-free fetal DNA (ccffDNA), originating from the placenta. The use of this DNA for non-invasive detection of fetal aneuploidies using massively parallel sequencing (MPS)-by-synthesis has been proven previously. Sequence performance may, however, depend on the MPS platform and therefore we have explored the possibility for multiplex MPS-by-ligation, using the Applied Biosystems SOLiD(?) 4 system. DNA isolated from plasma samples from 52 pregnant women, carrying normal or aneuploid fetuses, was sequenced in multiplex runs of 4, 8 or 16 samples simultaneously. The sequence reads were mapped to the human reference genome and quantified according to their genomic location. In case of a fetal aneuploidy, the number of reads of the aberrant chromosome is expected to be higher or lower than in normal reference samples. To statistically determine this, Z-scores per chromosome were calculated as described previously, with thresholds for aneuploidies set at > +3.0 and < -3.0 for chromosomal over- or underrepresentation, respectively. All samples from fetal aneuploidies yielded Z-scores outside the thresholds for the aberrant chromosomes, with no false negative or positive results. Full-blown fetal aneuploidies can thus be reliably detected in maternal plasma using a multiplex MPS-by-ligation approach. Furthermore, the results obtained with a sample from a pregnancy with 45,X in the cytotrophoblastic cell layer and 46,XX in the mesenchymal core cells show that ccffDNA originates from the cytotrophoblastic cell layer. Discrepancies between the genetic constitution of this cell layer and the fetus itself are well known, and therefore, care should be taken when translating results to the fetus itself.