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Ann Biol Clin (Paris). 2018 Oct 1;76(5):505-523. doi: 10.1684/abc.2018.1372.

Development of digital PCR molecular tests for clinical practice: principles, practical implementation and recommendations.

Author information

1
Sorbonne Université, Faculté de médecine et Institut universitaire en cancérologie ; Inserm UMR S 938, Centre de recherche de Saint-Antoine, Biologie et thérapeutiques du cancer ; Assistance Publique - Hôpitaux de Paris, Hôpitaux universitaires Pitié-Salpêtrière-Charles Foix, Service de biochimie endocrinienne et oncologique, Paris, France.
2
Service de génétique et biologie moléculaires, HUPC Hôpital Cochin, AP-HP, Paris, France.
3
Institut d'analyse génomique-imagenome, Labosud, Montpellier, France.
4
Fédération de génétique, CHI de Poissy-Saint-Germain en Laye, Poissy, France.
5
Sorbonne Université, Service d'hématologie biologique, Hôpital Saint-Antoine, AP-HP, Paris, France.
6
Service d'hématologie biologique, Hôpital Cochin, AP-HP, Paris, France.
7
Normandie Université, UniRouen, Inserm U1245, IRON group, Rouen University Hospital, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.
8
Plateforme de génomique-BMO et Centre de ressources biologiques, AMMICA, Inserm US23/CNRS UMS3655, Gustave Roussy, Villejuif, France.
9
Unité de génétique moléculaire - maladies héréditaires et oncologie, CHU Grenoble Alpes, Grenoble, France ; Institute for Advanced Biosciences, UGA/Inserm U1209/CNRS UMR5309, Grenoble, France.
10
Inserm UMR-S1147, CNRS SNC5014 ; Université Paris Descartes, Équipe labellisée Ligue nationale contre le cancer, Paris, France.
11
Assistance publique - Hôpitaux de Marseille, Service d'anatomie pathologique et neuropathologie, Marseille, France, ID Solutions, Grabels, France.

Abstract

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.

KEYWORDS:

digital PCR; dropplet; micro-compartiments; microfluidics; quantitative PCR; real-time PCR; recommendations

PMID:
30226193
DOI:
10.1684/abc.2018.1372
[Indexed for MEDLINE]

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