Does DNA extraction affect the specificity of a PCR method claiming the specific detectability of a genome-edited plant?

Under current EU legislation, genetically modified organisms (GMOs) and derived food and feed products must be authorized as GM food, feed, or seed and appropriate detection methods must be made available for use in official controls. A Real-Time PCR method has recently been published by Chhalliyil et al. claiming to be specific for the detection and identification of genome-edited oilseed rape (OSR) lines commercialized in North America.

Single and multi-laboratory validation of a droplet digital PCR method.

The authorisation of genetically modified food and feed in the EU is subject to the provision of evidence of safety and of the availability of reliable analytical methods. These methods represent an essential tool for official laboratories to enforce a harmonised market control. Here the validation of droplet digital PCR (dPCR) methods has been performed for studying if the performance and acceptance parameters set by EU and other international guidelines for the analysis of genetically modified organisms (GMO) in food and feed are suitable and achievable also with such methods.

Identification of single target taxon-specific reference assays for the most commonly genetically transformed crops using digital droplet PCR.

Knowledge of the number of DNA sequences targeted by the taxon-specific reference assays is essential for correct GM quantification and is key to the harmonisation of measurement results. In the present study droplet digital PCR (ddPCR) was used to determine the number of DNA target copies of taxon-specific assays validated for real-time PCR for the four main genetically modified (GM) crops. The transferability of experimental conditions from real-time PCR to ddPCR was also explored, as well as the effect of DNA digestion.

International ring trial for the validation of an event-specific Golden Rice 2 quantitative real-time polymerase chain reaction method.

This article describes the international validation of the quantitative real-time polymerase chain reaction (PCR) detection method for Golden Rice 2. The method consists of a taxon-specific assay amplifying a fragment of rice Phospholipase D α2 gene, and an event-specific assay designed on the 3' junction between transgenic insert and plant DNA. We validated the two assays independently, with absolute quantification, and in combination, with relative quantification, on DNA samples prepared in haploid genome equivalents.

Development, optimization, and single laboratory validation of an event-specific real-time PCR method for the detection and quantification of Golden Rice 2 using a novel taxon-specific assay.

In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness.

GMOMETHODS: the European Union database of reference methods for GMO analysis.

In order to provide reliable and harmonized information on methods for GMO (genetically modified organism) analysis we have published a database called "GMOMETHODS" that supplies information on PCR assays validated according to the principles and requirements of ISO 5725 and/or the International Union of Pure and Applied Chemistry protocol. In addition, the database contains methods that have been verified by the European Union Reference Laboratory for Genetically Modified Food and Feed in the context of compliance with an European Union legislative act. The web application provides search capabilities to retrieve primers and probes sequence information on the available methods.

Testing the interaction between analytical modules: an example with Roundup Ready soybean line GTS 40-3-2.

Background: The modular approach to analysis of genetically modified organisms (GMOs) relies on the independence of the modules combined (i.e. DNA extraction and GM quantification).

International collaborative study of the endogenous reference gene, sucrose phosphate synthase (SPS), used for qualitative and quantitative analysis of genetically modified rice.

One rice ( Oryza sativa ) gene, sucrose phosphate synthase (SPS), has been proven to be a suitable endogenous reference gene for genetically modified (GM) rice detection in a previous study. Herein are the reported results of an international collaborative ring trial for validation of the SPS gene as an endogenous reference gene and its optimized qualitative and quantitative polymerase chain reaction (PCR) systems. A total of 12 genetically modified organism (GMO) detection laboratories from seven countries participated in the ring trial and returned their results.