RASSF1A在无损性孕妇外周血浆中胎儿ABO基因分型中的运用

目的:探讨甲基化表观遗传标志RASSF1A在O型孕妇外周血浆中胎儿ABO基因分型中的运用,以无损性预测产前胎儿ABO血型。方法:首先提取O型孕妇外周血浆游离DNA,扩增SRY及甲基化RASSF1A作为胎儿DNA存在的标志;检测血浆游离DNA中B及非O预测胎儿ABO血型;并将基因分型结果与胎儿分娩后血清学结果进行比对,以评价该方法的准确性。结果:20例标本中,11例检测出SRY,检测结果与胎儿出生后性别相符;其余9例标本酶切后均扩增出RASSF1A,证实胎儿游离DNA存在。对血浆游离DNA进行ABO基因检测,20例标本中8例标本同时扩增出非O和B,提示为B血型;5例标本只扩增出非O,未扩增出B,提示为A型;7例标本均未扩增出非O及B,提示为O型。上述预测结果与胎儿出生后ABO血清学血型一致。结论:初步建立了基于RASSF1A的O型孕妇血浆中游离DNA中胎儿ABO基因的检测程序,可用于临床无损性产前胎儿ABO血型的预测。     

孕妇血浆中胎儿游离DNA在胎儿ABO血型鉴定中的应用

目的探索1种新的胎儿ABO血型的检测方法,为预防ABO血型系统新生儿溶血病提供可靠依据。方法收集医院2009年1～12月送检的O型孕妇34名,提取孕妇外周血血浆中胎儿游离DNA,利用聚合酶链反应———限制性片段长度多态性分型(PCR-RFLP)检测胎儿ABO血型。结果在34例标本中,32例实验成功,且与胎儿出生后的ABO血型一致,其中A型11例、B型15例、O型6例,2份标本扩增失败。结论利用孕妇外周血中胎儿游离DNA来检测胎儿ABO血型的方法,准确性高,安全性好,值得临床推广。     

利用孕妇血浆中游离DNA检测胎儿ABO基因型

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。     

从孕妇血浆中提取胎儿DNA鉴定胎儿RhCcEe血型

目的探讨利用孕妇血浆中游离胎儿DNA进行非创伤性产前诊断胎儿RhCcEe血型的方法。方法采用QIAamp DNA Kit抽提孕妇血浆DNA，利用SRY基因确认胎儿DNA的存在，通过PCR方法扩增30例孕妇血浆中胎儿DNA以检测胎儿RhCcEe基因，并对产前孕妇外周血和产后婴儿外周血进行RhCcEe血清学表型分析，回顾性评价胎儿基因分型结果的准确性。结果30例样本中，13例母子表型完全相同，17例存在区别。当母亲表型为RhCC、cc、EE、ee纯合子时，均成功扩增出母亲所缺少的c、C、e、E基因。结论本研究建立的非创伤性产前诊断胎儿RhCcEe基因型的方法是可行的，当母亲为纯合子时，血浆中胎儿RhCcEe基因分型具有临床意义，可用于新生儿溶血病的预防和诊断。     

PCR-SSP基因分型技术在ABO疑难血型定型中的应用

目的 研究聚合酶链式反应-特异性序列引物(PC-SSP)基因分型技术在ABO疑难血型鉴定中的应用.方法 收集8例来自广东省肇庆市无偿献血者的ABO疑难血型样本,采用正反定型实验、吸收放散试验等一系列血型血清学方法进行检测;提取基因组DNA,并用PCR-SSP基因分型技术,特异性扩增ABO基因片段,根据有无PCR产物以及产物长度,判断样本的基因型.结果 通过凝胶电泳检测PCR反应的特异性产物,判断样本1～6号ABO基因型为A/O型,样本7,8号为B/O型.8例疑难ABO基因分型结果与其血型的血清学分型结果吻合.结论 ABO疑难血型定型的解决是一个复杂的难题,而PCR-SSP基因分型是一种方便、快速、可靠的技术,对于解决ABO疑难血型鉴定非常有用.     

中国汉族人群ABO血型系统基因分型研究与应用

目的 研究中国汉族人群ABO基因多态性，并将基因分型技术用于解决临床输注中血型血清学难题：方法 快速盐析法提取外周血中的DNA，采用聚合酶链反应—序列特异性引物(PCR—SSP)基因方法对ABO血型定型，并且在吸收放散试验、唾液血型物质凝集抑制等血清学试验基础上，用PCR—SSP法扩增疑难ABO血型的等位基因：结果 在中国汉族符合Hardy—Weeinberg平衡的随机群体(260人)中，检出O1、B、A101(A267c)和A102／103(A467r)四种等位基因，其基因频率分别为、0.5827,0．1．846、0.0096、0.2231：在疑难血型鉴定中6份血清学定为A2的标本中，只有2例基因分型确定为A201O1，其余均为A102／A103O1型，在一家系疑难血型鉴定中，兄弟三人均为类孟买型，ABO基因分型分别为A102B、A102B、A102O1.结论 ABO RCR—SSP基因分型是一种方便、快速、可靠的技术，与血型血清学相比有着显著优势：研究发现中国汉族人群A2等位基因构成与国际报道的基因构成存在差异。     

中国汉族人群ABO血型系统基因分型研究与应用

目的研究中国汉族人群ABO基因多态性,并将基因分型技术用于解决临床输注中血型血清学难题.方法快速盐析法提取外周血中的DNA,采用聚合酶链反应-序列特异性引物(PCR-SSP)基因方法对ABO血型定型,并且在吸收放散试验、唾液血型物质凝集抑制等血清学试验基础上,用PCR-SSP法扩增疑难ABO血型的等位基因.结果在中国汉族符合Hardy-Weinberg平衡的随机群体(260人)中,检出O1、B、A101(A267c)和A102/103(A467r)四种等位基因,其基因频率分别为0.5827、0.1846、0.0096、0.2231.在疑难血型鉴定中6份血清学定为A2的标本中,只有2例基因分型确定为A201O1,其余均为A102/A103O1型,在一家系疑难血型鉴定中,兄弟三人均为类孟买型,ABO基因分型分别为A102B、A102B、A102O1.结论ABO RCR-SSP基因分型是一种方便、快速、可靠的技术,与血型血清学相比有着显著优势.研究发现中国汉族人群A2等位基因构成与国际报道的基因构成存在差异.     

实时荧光定量PCR检测孕妇血浆中胎儿RhCcEe基因

Rh血型系统在临床输血中的重要性仅次于ABO血型系统[1],Rh血型不合可引起溶血性输血反应及新生儿溶血病(HDN)[2].Lo等[2]研究证实孕妇血浆或血清中含有胎儿DNA,国外已用游离胎儿DNA开展了非创伤性产前诊断Rh基因的研究[4-7].我们曾用PCR扩增孕妇血浆中胎儿DNA,以检测胎儿RhCcEe基因[8].     

孕妇血浆游离DNA检测在母婴ABO血型不合诊断中的临床研究

目的 探索一种利用孕妇血浆中游离DNA检测胎儿ABO血型用于母婴ABO血型不合的无创性产前诊断方法.方法 用硅胶膜柱提取的方法从50例13～37孕周可疑母婴ABO血型不合的孕妇血浆中提取胎儿DNA,采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)法检测胎儿ABO血型.结果 50例样本中,检出32例,检出率64%(32/50),出生后血清学证实28例诊断正确,诊断正确率87.5%(28/32).结论 利用孕妇血浆中游离DNA检测胎儿ABO血型可行,对诊断和预防因母婴ABO血型不合所致溶血病的发生具有重要意义.     

PCR-SSP技术对羊水细胞ABO血型的基因鉴定

目的通过PCR-SSP基因技术检测胎儿羊水细胞ABO血型基因型,产前诊断胎儿ABO血型。方法选取了6名孕16 W以上的孕妇,抽取羊水细胞并进行分离,提取羊水细胞DNA,运用PCR-SSP技术分析其ABO血型基因型,并通过出生后的脐带血的血型鉴定进行确认。结果 6例羊水标本均通过PCR-SSP方法检测出了ABO血型的基因型;该6名胎儿的脐带血的ABO血型与羊水细胞的血型一致。结论 PCR-SSP技术可以准确地检测胎儿羊水细胞的ABO血型。     

Use of maternal plasma for non-invasive prenatal diagnosis of fetal ABO genotypes.

BACKGROUND: Measurement of free fetal DNA in maternal plasma opened a door for non-invasive prenatal diagnosis. Prenatal diagnosis of fetal ABO genotypes can provide a basis for the prevention and therapy of maternal-fetal incompatibility. We identified fetal ABO genotypes using fetal DNA in plasma from pregnant women with blood group O. The aim of the study was to investigate the accuracy and feasibility of this method. METHODS: A total of 105 blood group O women in middle or late pregnancy were enrolled. Fetal DNA in maternal plasma and genomic DNA in umbilical vein blood from newborns were extracted using a QIAamp DNA Blood Kit. DNA was amplified to identify ABO genotypes by PCR with sequence-specific primers (PCR-SSP). The genotype results were evaluated using serologic tests for ABO phenotyping. RESULTS: Using DNA from umbilical vein blood, ABO genotypes of 105 newborns were successfully identified by PCR-SSP. Using fetal DNA from maternal plasma, 88.6% (93/105) fetal ABO genotypes was correct; 12 false results were from 66 pregnant women with fetuses of type non-O. The accuracy in middle pregnancy was lower than that in late pregnancy, although the difference was not significant (0.05相似文献   

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.     

Hypermethylated RASSF1A in maternal plasma: A universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis

BACKGROUND: We recently demonstrated that the promoter of the RASSF1A gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. This methylation pattern allows the use of methylation-sensitive restriction enzyme digestion for detecting the placental-derived hypermethylated RASSF1A sequences in maternal plasma. METHODS: We performed real-time PCR after methylation-sensitive restriction enzyme digestion to detect placental-derived RASSF1A sequences in the plasma of 28 1st-trimester and 43 3rd-trimester pregnant women. We used maternal plasma to perform prenatal fetal rhesus D (RhD) blood group typing for 54 early-gestation RhD-negative women, with hypermethylated RASSF1A as the positive control for fetal DNA detection. RESULTS: Hypermethylated RASSF1A sequences were detectable in the plasma of all 71 pregnant women. The genotype of plasma RASSF1A after enzyme digestion was identical to the fetal genotype in each case, thus confirming its fetal origin. Nineteen of the 54 pregnant women undergoing prenatal fetal RhD genotyping showed undetectable RHD sequences in their plasma DNA samples. The fetal DNA control, RASSF1A, was not detectable in 4 of the 19 women. Subsequent chorionic villus sample analysis revealed that 2 of these 4 women with negative RHD and RASSF1A signals were in fact carrying RhD-positive fetuses. CONCLUSIONS: Hypermethylated RASSF1A is a universal marker for fetal DNA and is readily detectable in maternal plasma. When applied to prenatal RhD genotyping, this marker allows the detection of false-negative results caused by low fetal DNA concentrations in maternal plasma. This new marker can also be applied to many other prenatal diagnostic and monitoring scenarios.     

Noninvasive fetal RHD genotyping by maternal plasma with capillary electrophoresis

BACKGROUND: Recently, a more accurate and reliable screening test has been investigated for noninvasive prenatal fetal RHD genotyping from D- women. The objective of this study was to perform the new method of noninvasive fetal RHD genotyping with maternal plasma from D- women by use of capillary electrophoresis. STUDY DESIGN AND METHODS: Blood samples were obtained from 8 D+ and 8 D- nonpregnant donors and mixed to make test plasma samples. DNA was extracted and the appropriate conditions relating to the initial sample volume as well as polymerase chain reaction cycle numbers were analyzed to detect the RHD gene with RHD exon 10 primer. Blood samples were also obtained from 13 D- pregnant women ranging from the 12th to 39th weeks of gestation. The presence of the RHD gene and Y-chromosome-specific STR (Y-STR) derived from the fetus was analyzed. The results were compared with the D status of newborns. RESULTS: In samples from 12 D- pregnant women, the RHD gene was detected. In one sample, the RHD gene was not detected but Y-STR loci were demonstrated in this sample, indicating a D- male baby. The results of fetal genotyping were all in concordance with the postpartum samples by serologic tests on D as well as with the sex of newborns. CONCLUSION: Capillary electrophoresis can be used for the determination of fetal RHD status in D- women. This diagnostic method is useful for the noninvasive prenatal diagnosis of the fetal RHD genotyping from D- women.     

Large-scale pre-diagnosis study of fetal RHD genotyping by PCR on plasma DNA from RhD-negative pregnant women.

BACKGROUND: The routine prenatal determination of fetal RhD blood group would be very useful in the management of pregnancies in RhD-negative women, as up to 40% of these pregnancies bear a RhD-negative fetus. The fetal DNA present in maternal plasma offers an opportunity for risk-free prenatal diagnosis. AIM: This study focused on the feasibility and accuracy of large-scale RhD fetal diagnosis in non-immunized and anti-D immunized RhD-negative women. METHODS: Plasma DNA was extracted from 893 RhD-negative pregnant women and amplified in exons 7 and 10 of the RHD gene using conventional and real-time PCR. The results were then compared with the RHD fetal genotype determined on amniotic cells and/or the RhD phenotype of the red blood cells of the infants at birth. RESULTS: After exclusion of 42 samples from women exhibiting a nonfunctional or rearranged RHD gene, fetal RhD status was predicted with a 99.5% accuracy. A strategy is also proposed to avoid the small number of false-positive and -negative results. CONCLUSION: Fetal RHD genotyping from maternal plasma DNA in different clinical situations may be used with confidence.     

Fetal nucleic acids in maternal blood: the promises

Fetal DNA is present at an approximate mean fractional concentration of 10% in the plasma of pregnant women. The detection of paternally-inherited DNA sequences that are absent in the maternal genome, e.g., Y chromosomal sequences for fetal sexing and the RHD gene for blood group genotyping, is well established. The recent emergence of single molecule counting technologies, such as digital polymerase chain reaction and massively parallel sequencing has allowed circulating fetal DNA to be used for the non-invasive prenatal diagnosis of fetal chromosomal aneuploidies and monogenic diseases. With large scale clinical validation and further reduction in costs, it is expected that the analysis of circulating fetal DNA will play an increasingly important role in the future practice of prenatal diagnosis.     

Duffy血型基因定型及其用于HDN产前诊断的探讨

目的　探讨用分子生物学方法检测血液Duffy血型基因型及该方法应用于HDN产前检查的可行性。方法　采用聚合酶链反应 限制性酶切片短长度多态性 (PCR RFLP)方法检测Duffy基因型。结果　用本研究采用的方法检出了 90例中国辽宁地区汉族血液样品Duffy血型的 3种基因型 ;检测出丈夫为Fy(a+b - )型、本人为Fy(a -b +)型的孕妇羊水样品 1例 ,其Duffy基因型为FYAFYB。结论　本方法可准确检测血液及羊水样品的Duffy基因型 ,可用于Duffy血型不合引起的HDN的产前诊断     

汕头地区孕母产前血型抗体预测新生儿溶血病的研究

目的：探讨本地区ABO血型不合孕妇产前抗体效价检测在预测新生儿溶血病（HDN）中的意义。方法对536名血型不合的O型及Rh阴性孕妇在妊娠20周时进行血型不规则抗体筛选,抗体效价检测,效价≥64的定期（每4周）进行血型抗体效价复查检测,对新生儿出生后（0-7d）出现黄疸症状的抽静脉血3 ml进行血型血清学和溶血病三项检测。结果在536名孕妇血清中检出血型抗体303例,占56.5%,其中IgG抗体≥64者175例,占32.6%;,7名Rh阴性孕妇血清中产生抗D抗体3例,占0.6%;新生儿发生黄疸303例,诊断HDN 73例,占24.1%,接受治疗后均痊愈出院。结论汕头地区孕母产前血型抗体效价与新生儿溶血病密切相关,定期筛选与测定孕母体内血型抗体效价可预测HDN的发生,评估HDN的严重程度及提高HDN的治愈率。