Y chromosome detection by Real Time PCR and pyrophosphorolysis-activated polymerisation using free fetal DNA isolated from maternal plasma

2007 ◽  
Vol 27 (10) ◽  
pp. 932-937 ◽  
Author(s):  
Elles M. J. Boon ◽  
Hélène B. Schlecht ◽  
Peter Martin ◽  
Geoff Daniels ◽  
Rolf H. A. M. Vossen ◽  
...  
Keyword(s):  
Real Time ◽  
Y Chromosome ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Fetal Dna

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.

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.

scholarly journals Interlaboratory Comparison of Fetal Male DNA Detection from Common Maternal Plasma Samples by Real-Time PCR

2004 ◽  
Vol 50 (3) ◽  
pp. 516-521 ◽  
Author(s):  
Kirby L Johnson ◽  
Kimberly A Dukes ◽  
John Vidaver ◽  
Erik S LeShane ◽  
Idania Ramirez ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Genetic Diagnosis ◽  
Maternal Plasma ◽  
Standard Protocol ◽  
Plasma Samples ◽  
Entire Study ◽  
Fetal Dna ◽  
Plasma Fraction

Abstract Background: Analysis of fetal DNA from maternal plasma by PCR offers great potential for noninvasive prenatal genetic diagnosis. To further evaluate this potential, we developed and validated a standard protocol to determine whether fetal DNA sequences could be reproducibly amplified and measured across multiple laboratories in a common set of specimens. Methods: Each of five participating centers in a National Institute of Child Health and Human Development consortium collected 20 mL of peripheral blood from 20 pregnant women between 10 and 20 weeks of gestation. The plasma fraction was separated according to a common protocol, divided, and frozen in five aliquots. One aliquot was shipped to each participating laboratory, where DNA was extracted according to a standard protocol. All plasma samples (n = 100) were then analyzed blindly for the presence and quantity of total DNA (GAPDH) and male fetal DNA (SRY) by real-time PCR. Genomic DNA was isolated from female and male cells at one center, quantified, and shipped to the others to serve as calibrators for GAPDH and SRY, respectively. Results: The amplification of known quantities of DNA was consistent among all centers. The mean quantity of male DNA amplified from maternal plasma when the fetus was male ranged from 51 to 228 genome equivalents (GE)/mL. Qualitative concordance was found overall among centers. The sensitivity of the assay for detection of male DNA when the fetus was male varied from 31% to 97% among centers. Specificity was more consistent (93–100%) with only four false-positive results obtained across the entire study. Conclusions: All centers were able to consistently amplify frozen and shipped DNA. The PCR procedure used here is reliable and reproducible. Centers that extracted and amplified more DNA per milliliter of maternal plasma had superior sensitivities of Y chromosome sequence detection. The specificity of the assay was more consistent among centers. A robust and thoroughly optimized protocol for the extraction of DNA from maternal plasma is needed to make testing of fetal DNA in maternal plasma a clinically relevant analytical tool.

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 Non-invasive Fetal RHD and RHCE Genotyping Using Real-time PCR Testing of Maternal Plasma in RhD-negative Pregnancies

2005 ◽  
Vol 53 (3) ◽  
pp. 301-305 ◽  
Author(s):  
Ilona Hromadnikova ◽  
Lenka Vechetova ◽  
Klara Vesela ◽  
Blanka Benesova ◽  
Jindrich Doucha ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Real Time ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Plasma Samples ◽  
Maternal Circulation ◽  
Exon 2 ◽  
Non Invasive ◽  
Fetal Dna ◽  
Exon 10

We assessed the feasibility of fetal RHD and RHCE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analyzed 45 pregnant women in the 11th to 40th weeks of pregnancy and correlated the results with serological analysis of cord blood after delivery. Non-invasive prenatal fetal RHD exon 7, RHD exon 10, RHCE exon 2 (C allele), and RHCE exon 5 (E allele) genotyping analysis of maternal plasma samples was correctly performed in 45 out of 45 RhD-negative pregnant women delivering 24 RhD-, 17 RhC-, and 7 RhE-positive newborns. Detection of fetal RHD and the C and E alleles of RHCE gene from maternal plasma is highly accurate and enables implementation into clinical routine. We recommend performing fetal RHD and RHCE genotyping together with fetal sex determination in alloimmunized D-negative pregnancies at risk of hemolytic disease of the newborn. In case of D-negative fetus, amplification of another paternally inherited allele (SRY and/or RhC and/or RhE positivity) proves the presence of fetal DNA in maternal circulation.

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.

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