Title: Overview and recommendations for the application of digital PCR
Authors: PECORARO SVENBERBEN GILBERTBURNS M.CORBISIER PHILIPPEDE GIACOMO MARZIADE LOOSE MARCDAGAND EMILIEDOBNIK DAVIDERIKSSON RONNIEHOLST-JENSEN ARNEKAGKLI DAFNI MARIAKREYSA JOACHIMLIEVENS ANTOONMÄDE DIETRICHMAZZARA MARCOPATERNÒ ANNALISAPETERSEIL VERENASAVINI CRISTIANSOVOVÁ TEREZASOWA SLAWOMIRSPILSBERG BJØRN
Publisher: Publications Office of the European Union
Publication Year: 2019
JRC N°: JRC115736
ISBN: 978-92-76-00180-5 (online),978-92-76-00182-9 (print),978-92-76-07881-4 (ePub)
ISSN: 1831-9424 (online),1018-5593 (print)
Other Identifiers: EUR 29673 EN
OP KJ-NA-29673-EN-N (online),KJ-NA-29673-EN-C (print),KJ-NA-29673-EN-E (ePub)
URI: http://publications.jrc.ec.europa.eu/repository/handle/JRC115736
DOI: 10.2760/192883
10.2760/31034
10.2760/984242
Type: eBook
Abstract: The digital Polymerase Chain Reaction (dPCR), for the detection and absolute quantification of DNA, is a relatively new technique but its application in analytical laboratories is steadily increasing. In contrast to quantitative real-time PCR, DNA (fragments) can be quantified without the need for standard curves. Using dPCR, the PCR mix containing the (target) DNA is partitioned – depending on the device used – currently into a maximum of 10,000,000 small compartments with a volume as low as a few picoliters. These can be either physically distinct compartments on a chip (referred to as chamber-based digital PCR [cdPCR]), or these compartments correspond to water-in-oil droplets (referred to as droplet digital [ddPCR]). Common to both approaches, once PCR has been carried out simultaneously in all compartments/droplets, the number of positive and negative signals for each partition is counted by fluorescence measurement. With this technique, an absolute quantification of DNA copy numbers can be performed with high precision and trueness, even for very low DNA copy numbers. Furthermore, dPCR is considered less susceptible than qPCR to PCR inhibitory substances that can be co-extracted during DNA extraction from different sources. Digital PCR has already been applied in various fields, for example for the detection and quantification of GMOs, species (animals, plants), human diseases, food viruses and bacteria including pathogens. When establishing dPCR in a laboratory, different aspects have to be considered. These include, but are not limited to, the adjustment of the type of the PCR master mix used, optimised primer and probe concentrations and signal separation of positive and negative compartments. This document addresses these and other aspects and provides recommendations for the transfer of existing real-time PCR methods into a dPCR format.
JRC Directorate:Health, Consumers and Reference Materials

Files in This Item:
File Description SizeFormat 
kjna29673ene.epub2.71 MBeBookView/Open
overview_applicationdpcr_engl_online.pdf2.59 MBAdobe PDFView/Open


Items in repository are protected by copyright, with all rights reserved, unless otherwise indicated.