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1.
Ann Biol Clin (Paris) ; 76(5): 505-523, 2018 10 01.
Artigo em Francês | MEDLINE | ID: mdl-30226193

RESUMO

Digital PCR (dPCR) is a 3rd generation technology that complements traditional end-point PCR and real-time PCR. It was developed to overcome certain limitations of conventional amplification techniques, in particular for the detection of small amounts of nucleic acids and/or rare variants. This technology is in a full swing because of its high sensitivity and major applications in various domains such as oncology, transplantation or non-invasive prenatal testing. Consequently, PCRd also has great interest in many areas of medical biology, particularly for clinical applications aiming at detecting and quantifying specific genetic or epigenetic alterations of nucleic acids, even with specimens containing very low concentration of the nucleic acids of interest (e.g. liquid biopsies). However, this technique requires a good training of users and compliance with certain precautions. A lack in such a knowledge can lead to many errors in the conduct of the experiment and the interpretation of the results. In this review, we present the context in which this technology has emerged by describing in particular its principle and the main factors that can influence the quality of the analysis. Then, we propose a number of practical recommendations for the implementation of a test based on dPCR in clinical laboratories with an eye on quality requirements.


Assuntos
Técnicas de Laboratório Clínico/métodos , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Técnicas de Laboratório Clínico/normas , Feminino , Humanos , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase/normas , Guias de Prática Clínica como Assunto , Gravidez , Diagnóstico Pré-Natal/métodos , Diagnóstico Pré-Natal/normas , Reação em Cadeia da Polimerase em Tempo Real , Processamento de Sinais Assistido por Computador
2.
PLoS One ; 11(5): e0155009, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27167625

RESUMO

OBJECTIVE: NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. METHOD: After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. RESULTS: The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample's trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups. CONCLUSION: Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic setting and compatible with ethical concerns regarding access to nucleotide sequence information. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women.


Assuntos
Síndrome de Down/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Diagnóstico Pré-Natal/métodos , Cromossomos Humanos Par 21/genética , DNA/sangue , Sondas de DNA/metabolismo , Síndrome de Down/sangue , Síndrome de Down/genética , Humanos , Curva ROC , Reprodutibilidade dos Testes
3.
Mol Diagn ; 8(1): 23-31, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15230639

RESUMO

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.


Assuntos
DNA/sangue , DNA/genética , Sangue Fetal/imunologia , Sistema do Grupo Sanguíneo Rh-Hr/genética , Sequência de Bases , Erros de Diagnóstico , Eritroblastose Fetal/sangue , Eritroblastose Fetal/diagnóstico , Eritroblastose Fetal/genética , Éxons , Feminino , Genótipo , Humanos , Recém-Nascido , Íntrons , Masculino , Reação em Cadeia da Polimerase , Gravidez , Diagnóstico Pré-Natal
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