Non-invasive fetal ABO genotyping in maternal plasma using real-time PCR

2015 ◽  
Vol 53 (12) ◽  
Author(s):  
Wenqian Song ◽  
Shihang Zhou ◽  
Linnan Shao ◽  
Ni Wang ◽  
Lingzi Pan ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Forensic Analysis ◽  
Igg Antibody ◽  
Non Invasive ◽  
Abo Incompatibility ◽  
Cell Free Fetal Dna ◽  
Methylation Sensitive Restriction Enzyme ◽  
Maternal Igg

AbstractFetal-maternal ABO incompatibility is a frequent cause of hemolytic disease of the fetus and newborn (HDFN). The routine serological testing of maternal IgG antibody level to predict HDFN shows low reliability. Non-invasive fetal ABO genotyping could provide a new avenue for predicting ABO-HDFN in early pregnancy. The aim of our study is to investigate the feasibility of fetal ABO genotyping in maternal plasma with real-time PCR.Plasma samples were collected from a total of 73 blood group O pregnant women between 12 and 25 weeks of gestation, and then DNA was extracted from the maternal plasma containing cell-free fetal DNA (cffDNA). TaqMan-based real-time PCR was performed after methylation-sensitive restriction enzyme to detect hypermethylatedThe fetalWe have developed a rapid and reliable protocol for fetal ABO genotyping in maternal plasma using real-time PCR. This protocol is suitable for routine prenatal diagnose of HDFN and forensic analysis.

Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma

2015 ◽  
Vol 53 (8) ◽  
Author(s):  
Sherry Sze Yee Ho ◽  
Angela Barrett ◽  
Henna Thadani ◽  
Cecille Laureano Asibal ◽  
Evelyn Siew-Chuan Koay ◽  
...  
Keyword(s):  
Real Time ◽  
Y Chromosome ◽  
Real Time Pcr ◽  
Genetic Material ◽  
Maternal Plasma ◽  
Fetal Sex ◽  
Female Fetus ◽  
Non Invasive ◽  
Fetal Dna ◽  
Cell Free Fetal Dna

AbstractPrenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA.Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence,Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples.We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

scholarly journals Two Reliable Methodical Approaches for Non-Invasive RHD Genotyping of a Fetus from Maternal Plasma

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 564
Author(s):  
Jana Bohmova ◽  
Marek Lubusky ◽  
Iva Holuskova ◽  
Martina Studnickova ◽  
Romana Kratochvilova ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Real Time ◽  
Real Time Pcr ◽  
Dna Analysis ◽  
Methodological Approach ◽  
Maternal Plasma ◽  
Buccal Swab ◽  
Non Invasive ◽  
Pcr Method ◽  
Parallel Reactions

Noninvasive fetal RHD genotyping is an important tool for predicting RhD incompatibility between a pregnant woman and a fetus. This study aimed to assess a methodological approach other than the commonly used one for noninvasive fetal RHD genotyping on a representative set of RhD-negative pregnant women. The methodology must be accurate, reliable, and broadly available for implementation into routine clinical practice. A total of 337 RhD-negative pregnant women from the Czech Republic region were tested in this study. The fetal RHD genotype was assessed using two methods: real-time PCR and endpoint quantitative fluorescent (QF) PCR. We used exon-7-specific primers from the RHD gene, along with internal controls. Plasma samples were analyzed and measured in four/two parallel reactions to determine the accuracy of the RHD genotyping. The RHD genotype was verified using DNA analysis from a newborn buccal swab. Both methods showed an excellent ability to predict the RHD genotype. Real-time PCR achieved its greatest accuracy of 98.6% (97.1% sensitivity and 100% specificity (95% CI)) if all four PCRs were positive/negative. The QF PCR method also achieved its greatest accuracy of 99.4% (100% sensitivity and 98.6% specificity (95% CI)) if all the measurements were positive/negative. Both real-time PCR and QF PCR were reliable methods for precisely assessing the fetal RHD allele from the plasma of RhD-negative pregnant women.

Non-Invasive Fetal RHD Exon 7 and Exon 10 Genotyping Using Real-Time PCR Testing of Fetal DNA in Maternal Plasma

2005 ◽  
Vol 20 (4) ◽  
pp. 275-280 ◽  
Author(s):  
Ilona Hromadnikova ◽  
Lenka Vechetova ◽  
Klara Vesela ◽  
Blanka Benesova ◽  
Jindrich Doucha ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Non Invasive ◽  
Fetal Dna ◽  
Exon 10

An Effective Method for Detecting Y-chromosome Specific Sequences of Circulating Fetal DNA in Maternal Plasma During the First-trimester

2019 ◽  
Author(s):  
Najmeh Davoodian ◽  
Ali Kadivar ◽  
Heidar Heidari Khoie ◽  
Sima Hematian Khayat ◽  
Mahboobeh Heidari Nasirabadi
Keyword(s):  
Prenatal Diagnosis ◽  
Pregnant Women ◽  
Real Time ◽  
Y Chromosome ◽  
Maternal Plasma ◽  
Fetal Sex ◽  
Non Invasive ◽  
Fetal Dna ◽  
Fetal Gender ◽  
Cell Free Fetal Dna

Background and Aims: New advances in the use of cell-free fetal DNA (cffDNA) in maternal plasma of pregnant women has provided the possibility of applying cffDNA in prenatal diagnosis as a non-invasive method. One of the applications of prenatal diagnosis is fetal gender determination. Early prenatal determination of fetal sex is required for pregnant women at risk of X-linked and some endocrine diseases. The present study was carried out to perform an efficient polymerase chain reaction (PCR) method in order to improve sensitivity, specificity and accuracy of non-invasive fetal gender detection using fetal DNA in maternal plasma during 8th -12th weeks of pregnancy. Materials and Methods: Thirty-five pregnant women with 8 to 12 weeks of pregnancy were selected for prenatal fetal sex determination. Maternal peripheral blood was collected and cffDNA was extracted from 3-ml of maternal plasma. Two multi copy Y-chromosome-specific region (DYS and DAZ) and a single copy gene (SRY) were amplified by real-time quantitative PCR. Amplification was labeled as positive, negative, or inconclusive according to a stringent algorithm. Results: Using this method, the sensitivity and specificity of the real-time PCR assay was 100% and 93.8% for prenatal fetal sex detection, respectively. Conclusions: It is concluded that fetal sex can be determined with a high level of accuracy by our algorithm, after 8 weeks of gestation with cffDNA analysis.

Replicate real-time PCR testing of DNA in maternal plasma increases the sensitivity of non-invasive fetal sex determination

2003 ◽  
Vol 23 (3) ◽  
pp. 235-238 ◽  
Author(s):  
Ilona Hromadnikova ◽  
Bela Houbova ◽  
Dana Hridelova ◽  
Sona Voslarova ◽  
Josef Kofer ◽  
...  
Keyword(s):  
Sex Determination ◽  
Real Time ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Fetal Sex ◽  
Non Invasive

scholarly journals Risk Minimization of Hemolytic Disease of the Fetus and Newborn Using Droplet Digital PCR Method for Accurate Fetal Genotype Assessment of RHD, KEL, and RHCE from Cell-Free Fetal DNA of Maternal Plasma

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 803
Author(s):  
Radek Vodicka ◽  
Jana Bohmova ◽  
Iva Holuskova ◽  
Eva Krejcirikova ◽  
Martin Prochazka ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Maternal Plasma ◽  
Nucleotide Polymorphisms ◽  
Risk Minimization ◽  
Plasma Dna ◽  
Hemolytic Disease ◽  
Cell Free Fetal Dna ◽  
Clinically Significant

The molecular pathology of hemolytic disease of the fetus and newborn (HDFN) is determined by different RHD, RHCE, and KEL genotypes and by blood group incompatibility between the mother and fetus that is caused by erythrocyte antigen presence/absence on the cell surface. In the Czech Republic, clinically significant antierythrocyte alloantibodies include anti-D, anti-K, anti C/c, and anti-E. Deletion of the RHD gene and then three single nucleotide polymorphisms in the RHCE and KEL genes (rs676785, rs609320, and rs8176058) are the most common. The aim of this study is to develop effective and precise monitoring of fetal genotypes from maternal plasma of these polymorphisms using droplet digital (dd)PCR. Fifty-three plasma DNA samples (from 10 to 18 weeks of gestation) were analyzed (10 RHD, 33 RHCE, and 10 KEL). The ddPCR methodology was validated on the basis of the already elaborated and established method of minisequencing and real-time PCR and with newborn phenotype confirmation. The results of ddPCR were in 100% agreement with minisequencing and real-time PCR and also with newborn phenotype. ddPCR can fully replace the reliable but more time-consuming method of minisequencing and real-time PCR RHD examination. Accurate and rapid noninvasive fetal genotyping minimizes the possibility of HDFN developing.

Non-invasive foetalRHDgenotyping via real-time PCR of foetal DNA from Chinese RhD-negative maternal plasma

2009 ◽  
Vol 39 (7) ◽  
pp. 607-617 ◽  
Author(s):  
X. D. Wang ◽  
B. L. Wang ◽  
S. L. Ye ◽  
Y. Q. Liao ◽  
L. F. Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Non Invasive

scholarly journals Microfluidics Digital PCR Reveals a Higher than Expected Fraction of Fetal DNA in Maternal Plasma

2008 ◽  
Vol 54 (10) ◽  
pp. 1664-1672 ◽  
Author(s):  
Fiona M F Lun ◽  
Rossa W K Chiu ◽  
K C Allen Chan ◽  
Tak Yeung Leung ◽  
Tze Kin Lau ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Maternal Plasma ◽  
Clinical Sensitivity ◽  
Fetal Dna ◽  
Fractional Concentration ◽  
Cell Free Fetal Dna ◽  
Diagnostic Applications ◽  
Circulating Fetal Dna

Abstract Background: The precise measurement of cell-free fetal DNA in maternal plasma facilitates noninvasive prenatal diagnosis of fetal chromosomal aneuploidies and other applications. We tested the hypothesis that microfluidics digital PCR, in which individual fetal-DNA molecules are counted, could enhance the precision of measuring circulating fetal DNA. Methods: We first determined whether microfluidics digital PCR, real-time PCR, and mass spectrometry produced different estimates of male-DNA concentrations in artificial mixtures of male and female DNA. We then focused on comparing the imprecision of microfluidics digital PCR with that of a well-established nondigital PCR assay for measuring male fetal DNA in maternal plasma. Results: Of the tested platforms, microfluidics digital PCR demonstrated the least quantitative bias for measuring the fractional concentration of male DNA. This assay had a lower imprecision and higher clinical sensitivity compared with nondigital real-time PCR. With the ZFY/ZFX assay on the microfluidics digital PCR platform, the median fractional concentration of fetal DNA in maternal plasma was ≥2 times higher for all 3 trimesters of pregnancy than previously reported. Conclusions: Microfluidics digital PCR represents an improvement over previous methods for quantifying fetal DNA in maternal plasma, enabling diagnostic and research applications requiring precise quantification. This approach may also impact other diagnostic applications of plasma nucleic acids, e.g., in oncology and transplantation.

scholarly journals Detection of Restriction Enzyme–Digested Target DNA by PCR Amplification Using a Stem-Loop Primer: Application to the Detection of Hypomethylated Fetal DNA in Maternal Plasma

2007 ◽  
Vol 53 (11) ◽  
pp. 1906-1914 ◽  
Author(s):  
Yu K Tong ◽  
Rossa WK Chiu ◽  
Tak Y Leung ◽  
Chunming Ding ◽  
Tze K Lau ◽  
...  
Keyword(s):  
Real Time ◽  
Restriction Enzyme ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Plasma Samples ◽  
Dna Molecules ◽  
Stem Loop ◽  
Loop Primer ◽  
Fetal Dna ◽  
Cell Free Fetal Dna

Abstract Background: The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis and monitoring. Among the fetal markers that have been described, methylation markers are sex and polymorphism independent. Methylation-sensitive restriction endonucleases are commonly used to digest hypomethylated DNA molecules, and the hypermethylated molecules remain intact for detection. The positive detection of the cleaved hypomethylated molecules would be useful for certain targets but has not been reported. Methods: The use of a stem-loop primer in microRNA detection has previously been described. In this study, DNA assays were designed and performed on maternal plasma, which contained the hypomethylated placental serpin peptidase inhibitor, clade B (ovalbumin), member 5 (SERPINB5; maspin) gene in an excess background of hypermethylated maternal SERPINB5. Detection of the enzyme-digested placenta-derived hypomethylated SERPINB5 molecules was achieved by performing stem-loop extension followed by real-time PCR on maternal plasma. The placental origin of the stem-loop–extended SERPINB5 molecules was confirmed by genotyping. Results: From the real-time PCR results on maternal plasma, stem-loop–extended SERPINB5 promoter sequences were detectable in all 11 enzyme-digested predelivery maternal plasma samples. Postpartum clearance was demonstrated. In 9 cases in which the fetal and maternal SERPINB5 genotypes were distinguishable, the placental-specific genotypes were detected in all predelivery maternal plasma samples. Conclusion: Detection of restriction enzyme-digested hypomethylated placental DNA molecules in maternal plasma by the use of a stem-loop primer represents a novel approach in fetal epigenetic marker detection. The analytical approach may also be generally applicable to the detection of restriction enzyme-digested nucleic acid fragments.

scholarly journals DETERMINATION OF FETAL RHESUS D STATUS BY MATERNAL PLASMA DNA ANALYSIS

2013 ◽  
Vol 16 (2) ◽  
pp. 33-38 ◽  
Author(s):  
A. Aykut ◽  
H. Onay ◽  
C. Gunduz ◽  
F. Ozkinay ◽  
O. Cogulu ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Real Time ◽  
Real Time Pcr ◽  
Dna Analysis ◽  
Maternal Plasma ◽  
Clinical Samples ◽  
Plasma Dna ◽  
Fetal Sex ◽  
Cell Free Fetal Dna

ABSTRACT In this study, we assessed the feasibility of fetal RhD genotyping by analysis of cell-free fetal DNA(cffDNA) extracted from plasma samples of Rhesus (Rh) D-negative pregnant women by using real-time polymerase chain reaction (PCR). Fetal genotyping was performed on 30 RhD-negative women between 9 and 39 weeks of gestation who were referred to us for invasive testing [amniocentesis/ chorionic villi sampling (CVS)]. The fetal RHD genotype was determined based on real-time PCR method. Exons 7 and 10 of the RHD and SRY genes were targeted. Among the pregnant women, 12 were carrying male and 17 were carrying female fetuses. Out of 29 pregnant women, 21 had RhD-positive and nine had RhD-negative fetuses. One sample )case 12, whose blood group was found to be AB Rh [+] (was excluded due to controversial results from repeated serological analyses. All prenatal results were in concordance with postnatal RhD status and fetal sex without false- positive or -negative results. Performing real-time PCR on cffDNA showed accurate, efficient and reliable results, allowing rapid and high throughput non invasive determination of fetal sex and RhD status in clinical samples.

Sign in / Sign up

Export Citation Format

Share Document