scholarly journals Detection of Increased Amounts of Cell-Free Fetal DNA with Short PCR Amplicons

2010 ◽  
Vol 56 (1) ◽  
pp. 136-138 ◽  
Author(s):  
Aleksandra Sikora ◽  
Bernhard G Zimmermann ◽  
Corinne Rusterholz ◽  
Daniella Birri ◽  
Varaprasad Kolla ◽  
...  
Keyword(s):  
Real Time ◽  
Digital Pcr ◽  
Maternal Plasma ◽  
Qpcr Assay ◽  
Detection Capability ◽  
Real Time Quantitative Pcr ◽  
Fetal Dna ◽  
Cell Free Fetal Dna ◽  
Amplicon Size ◽  
Real Time Qpcr

Abstract Aim: A digital PCR approach has recently been suggested to detect greater amounts of cell-free fetal DNA in maternal plasma than conventional real-time quantitative PCR (qPCR). Because the digital qPCR approach uses shorter PCR amplicons than the real-time qPCR assay, we investigated whether a real-time qPCR assay appropriately modified for such short amplicons would improve the detection of cell-free fetal DNA. Method: We developed a novel universal-template (UT) real-time qPCR assay that was specific for the DYS14 sequence on Y chromosome and had a short amplicon size of 50 bp. We examined this “short” assay with 50 maternal plasma samples and compared the results with those for a conventional real-time qPCR assay of the same locus but with a longer amplicon (84 bp). Results: Qualitatively, both assays detected male cell-free fetal DNA with the same specificity and detection capability. Quantitatively, however, the new UT real-time qPCR assay for shorter amplicons detected, on average, almost 1.6-fold more cell-free fetal DNA than the conventional real-time qPCR assay with longer amplicons. Conclusions: The use of short PCR amplicons improves the detection of cell-free fetal DNA. This feature may prove useful in attempts to detect cell-free fetal DNA under conditions in which the amount of template is low, such as in samples obtained early in pregnancy.

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 Increased Fetal DNA Concentrations in the Plasma of Pregnant Women Carrying Fetuses with Trisomy 21

1999 ◽  
Vol 45 (10) ◽  
pp. 1747-1751 ◽  
Author(s):  
YM Dennis Lo ◽  
Tze K Lau ◽  
Jun Zhang ◽  
Tse N Leung ◽  
Allan MZ Chang ◽  
...  
Keyword(s):  
Hong Kong ◽  
Real Time ◽  
Trisomy 21 ◽  
Clinical Investigation ◽  
Maternal Plasma ◽  
Pcr Analysis ◽  
Fetal Dna ◽  
Prenatal Diagnostic ◽  
Cell Free Fetal Dna ◽  
Circulating Fetal Dna

Abstract Background: The recent discovery of the presence of circulating cell-free fetal DNA in maternal plasma opens up new prenatal diagnostic applications and provides new avenues for clinical investigation. It is of research and potential diagnostic interest to determine whether fetal trisomy 21 may be associated with quantitative abnormalities of circulating fetal DNA in maternal plasma. Methods: Maternal plasma samples were prospectively collected from two centers situated in Hong Kong and Boston. Samples collected from Boston consisted of 7 women carrying male trisomy 21 fetuses, 19 carrying euploid male fetuses, and 13 carrying female fetuses. Samples collected from Hong Kong consisted of 6 women carrying male trisomy 21 fetuses, 18 carrying euploid male fetuses, and 10 carrying female fetuses. Male fetal DNA in maternal plasma was measured using real-time quantitative Y-chromosomal PCR. Results: For patients recruited from Boston, the median circulating fetal DNA concentrations in women carrying trisomy 21 and euploid male fetuses were 46.0 genome-equivalents/mL and 23.3 genome-equivalents/mL, respectively (P = 0.028). For patients recruited from Hong Kong, the median circulating fetal DNA concentrations in women carrying trisomy 21 and euploid male fetuses were 48.2 genome-equivalents/mL and 16.3 genome-equivalents/mL, respectively (P = 0.026). None of the samples from women carrying female fetuses had detectable Y-chromosomal signals. Conclusions: Abnormally high concentrations of circulating fetal DNA are found in a proportion of women carrying fetuses with trisomy 21. The robustness and reproducibility of real-time PCR analysis of maternal plasma makes it a valuable tool for cross-institutional collaboration involving centers located in different parts of the world.

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.

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 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.

Optimized Real-Time Quantitative PCR Measurement of Male Fetal DNA in Maternal Plasma

2006 ◽  
Vol 1075 (1) ◽  
pp. 347-349 ◽  
Author(s):  
B. G ZIMMERMANN ◽  
W. HOLZGREVE ◽  
N. AVENT ◽  
S. HAHN
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Maternal Plasma ◽  
Real Time Quantitative Pcr ◽  
Fetal Dna

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 Optimized Real-Time Quantitative PCR Measurement of Male Fetal DNA in Maternal Plasma

2005 ◽  
Vol 51 (9) ◽  
pp. 1598-1604 ◽  
Author(s):  
Bernhard Zimmermann ◽  
Ahmad El-Sheikhah ◽  
Kypros Nicolaides ◽  
Wolfgang Holzgreve ◽  
Sinuhe Hahn
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Pcr Amplification ◽  
First Trimester ◽  
Single Copy ◽  
Maternal Plasma ◽  
Probit Regression ◽  
Real Time Quantitative Pcr ◽  
Fetal Dna ◽  
First Trimester Of Pregnancy

Abstract Background: Circulating fetal DNA (cfDNA) in maternal plasma has been measured to investigate its possible relationship with pregnancy-related disorders, including fetal trisomy 21 and preeclampsia. The circulating concentrations of single-copy fetal genes, however, are close to the detection limits of PCR methods. Methods: We optimized a protocol for the real-time quantitative PCR amplification of the multicopy sequence DYS14 on the Y-chromosome. This was compared with an established real-time PCR assay for the single-copy SRY gene. Results: By probit regression analysis, the measurements of male DNA by the DYS14 assay had a 10-fold lower detection limit (0.4 genome equivalents) than did measurements of SRY. For plasma samples from women in the first trimester of pregnancy, imprecision (CV) was 2%–22% when amplifying DYS14 compared with 26%–140% for SRY. Conclusions: The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy cannot be measured precisely when targeting single-copy sequences. Better results are obtained by amplifying a sequence that is present in multiple copies per male genome.

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