Overview and recommendations for the application of digital PCR

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.F.5-Food and Feed Complianc

Use of Novel Techniques in Plant Breeding and Practical Consequences Concerning Detection, Traceability, Labeling, and Risk Assessment

Genetically modified plants (GMP) are regulated by comprehensive EU legislation. Cisgenesis, intragenesis, oligo-directed mutagenesis (ODM), zinc-finger nucleases (ZFN), and agroinfiltration were examined concerning detection, traceability, labeling, and risk assessment. Similar transformation methods -- as in transgenesis using genetic elements from same or cross-compatible plant species -- characterize cisgenesis and intragenesis. Targeting the genome with ODM or ZFN minimizes unintended effects, but current data indicate limited efficiency and specificity; modifications are similar to those occurring during traditional plant breeding. The characteristics of plants produced by these techniques affect detection in the supply chains. Detection is possible when the target site is known, except for agroinfiltration that aims at transient expression of a gene/trait. The basis to assess potential risks arising from relevant plants and derived food and feed is similar to that of GMP. Depending on the specifics of the plant under investigation, data requirements for regulators may be reconsidered case-by-case