Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR

Sillence, Kelly A.; Roberts, Llinos A.; Hollands, Heidi; Thompson, Hannah P.; Kiernan, Michele; Madgett, Tracey E.; Welch, C.; Avent, Neil D.

doi:10.1373/clinchem.2015.239137

Cited by 49 publications

(33 citation statements)

References 39 publications

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“…As a result, dPCR can detect small fold changes in gene expression much more accurately than qPCR. The dPCR has been proposed for the prenatal detection of single‐gene disorders, but it may also be useful for the development of circulating RNA biomarkers . However, while qPCR is an established technique in the majority of clinical laboratories, dPCR remains to be validated for routine clinical use, and therefore, its diagnostic use is presently limited.…”

Section: Contemporary Approaches To Measure Circulating Placenta Rnamentioning

confidence: 99%

Measuring circulating placental RNAs to non-invasively assess the placental transcriptome and to predict pregnancy complications

2016

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Circulating nucleic acids have revolutionized prenatal diagnosis in the last decade, allowing non-invasive screening for single gene or chromosomal defects using a single sample of maternal blood. In addition to DNAs, RNAs from the placenta are released into the maternal blood from early in pregnancy and may reflect changes in gene expression occurring within the placenta. Measuring circulating RNA may therefore provide insights into the placental transcriptome without the need for invasive testing. Combined with advances in next-generation sequencing and molecular analyses, it may be possible to measure circulating RNA to improve our understanding of placental pathology and develop novel non-invasive biomarkers for pregnancy complications and monitoring high-risk pregnancies. This review summarizes the current technologies available and the studies that have measured circulating placental RNA to predict and/or monitor pregnancies complicated by preeclampsia, fetal growth restriction, preterm birth, early pregnancy complications, invasive placentation and twin-twin transfusion syndrome. Prospective cohort studies are now required to validate these findings to determine the clinical applicability of measuring circulating placental RNA to develop novel biomarkers for a wide spectrum of pregnancy complications.

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Section: Contemporary Approaches To Measure Circulating Placenta Rnamentioning

confidence: 99%

Measuring circulating placental RNAs to non-invasively assess the placental transcriptome and to predict pregnancy complications

2016

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“…A number of studies on ddPCR show high sensitivity of the method and high precision for absolute quantification of DNA targets, for example in cancer [5–8], hepatitis B virus [9, 10], cytomegalovirus[11, 12], Chlamydia trachomatis [13, 14], Babesia microti , and Babesia duncani [15]. Comparison of ddPCR assays with real-time PCR assays showed that ddPCR assays had a higher sensitivity than real-time PCR assays [16, 17], but this was not confirmed in some other studies [11, 18]. Real-time PCR assays are widely used for detection and relative quantification of Plasmodium parasites.…”

Section: Introductionmentioning

confidence: 99%

Four human Plasmodium species quantification using droplet digital PCR

et al. 2017

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Droplet digital polymerase chain reaction (ddPCR) is a partial PCR based on water-oil emulsion droplet technology. It is a highly sensitive method for detecting and delineating minor alleles from complex backgrounds and provides absolute quantification of DNA targets. The ddPCR technology has been applied for detection of many pathogens. Here the sensitive assay utilizing ddPCR for detection and quantification of Plasmodium species was investigated. The assay was developed for two levels of detection, genus specific for all Plasmodium species and for specific Plasmodium species detection. The ddPCR assay was developed based on primers and probes specific to the Plasmodium genus 18S rRNA gene. Using ddPCR for ultra-sensitive P. falciparum assessment, the lower level of detection from concentrated DNA obtained from a high volume (1 mL) blood sample was 11 parasites/mL. For species identification, in particular for samples with mixed infections, a duplex reaction was developed for detection and quantification P. falciparum/ P. vivax and P. malariae/ P. ovale. Amplification of each Plasmodium species in the duplex reaction showed equal sensitivity to singleplex single species detection. The duplex ddPCR assay had higher sensitivity to identify minor species in 32 subpatent parasitaemia samples from Cambodia, and performed better than real-time PCR. The ddPCR assay shows high sensitivity to assess very low parasitaemia of all human Plasmodium species. This provides a useful research tool for studying the role of the asymptomatic parasite reservoir for transmission in regions aiming for malaria elimination.

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“…RCD can detect fetal trisomy 21 in maternal plasma [33]. RCD can also determine the sex of the fetus from cffDNA [34]. As a proof of concept, RMD detects single nucleotide changes or small deletions in HBB ( β-globin ; HbE, ( HBB(E26K) ) and CD41/42 allele (a 4 nucleotide deletion at codons 41 and 42)) associated with β-thalassemia [35].…”

Section: Applications Of Digital Pcr To Pediatric Geneticsmentioning

confidence: 99%

“…Standard genetic screen can identify RhD + pregnancies in mothers completely lacking RHD but not in those mothers harboring intragenic mutations in RHD . dPCR can detect paternally-inherited, fetal RHD in maternal plasma [34], [45]. dPCR can accurately detect maternal RHD variants like RHD(IVS3 + 1G > A) in the presence of cffDNA containing paternally-inherited RHD [46].…”

Section: Applications Of Digital Pcr To Pediatric Geneticsmentioning

confidence: 99%

“…The increased sensitivity of dPCR at detecting low levels of target DNA within a complex mixture of DNA has been demonstrated by detecting subclonal SETBP1 point mutations in patients with JMML, a low frequency somatic GNAQ(R183Q) mutation in patients with Sturge-Weber syndrome [28] and a GNAS1(R201C) mutation within some tissues of McCune-Albright syndrome post-mortem case [27], [29]. In cffDNA, dPCR is more sensitive than standard qPCR in detecting fetal RHD as well as the sex of the fetus in maternal plasma [34], [45]. dPCR, therefore, is better capable of analyzing complex mixtures of DNA to detect rare locus events than qPCR.…”

Section: Comparison Of Digital Pcr To Other Techniquesmentioning

confidence: 99%

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Applicability of digital PCR to the investigation of pediatric-onset genetic disorders

Butchbach

2016

Biomolecular Detection and Quantification

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Early-onset rare diseases have a strong impact on child healthcare even though the incidence of each of these diseases is relatively low. In order to better manage the care of these children, it is imperative to quickly diagnose the molecular bases for these disorders as well as to develop technologies with prognostic potential. Digital PCR (dPCR) is well suited for this role by providing an absolute quantification of the target DNA within a sample. This review illustrates how dPCR can be used to identify genes associated with pediatric-onset disorders, to identify copy number status of important disease-causing genes and variants and to quantify modifier genes. It is also a powerful technology to track changes in genomic biomarkers with disease progression. Based on its capability to accurately and reliably detect genomic alterations with high sensitivity and a large dynamic detection range, dPCR has the potential to become the tool of choice for the verification of pediatric disease-associated mutations identified by next generation sequencing, copy number determination and noninvasive prenatal screening.

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Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR

Cited by 49 publications

References 39 publications

Measuring circulating placental RNAs to non-invasively assess the placental transcriptome and to predict pregnancy complications

Measuring circulating placental RNAs to non-invasively assess the placental transcriptome and to predict pregnancy complications

Four human Plasmodium species quantification using droplet digital PCR

Applicability of digital PCR to the investigation of pediatric-onset genetic disorders

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