Description of the subject. This paper discusses the influence of amplicon length on real-time PCR results. Objectives. The aim of the experiments was to show that amplicon size has an influence on detection. Method. Tests were performed on genomic and plasmid DNA. Double-dye probes and SYBR® Green were used for detection by real-time PCR. Primers were selected in order to produce fragments with increasing sizes. Experiments dealt with two targets: an endogenous target for soybean (part of the lectin gene) and a transgenic target (junction P35S-CTP of the MON40-3-2 soybean). Results. The results show that the kinetics of amplification curves evolve as a function of amplicon length, and smaller amplicons yield a higher level of fluorescence for the plateau phase. DNA degradation within the sample as well as the principles of fluorescence acquisition as a function of the chemistry used can also be factors. Conclusions. It was experimentally shown that the observed effect is linked to the suboptimal elongation temperature used in real-time PCR. Detection using SYBR® Green is less impacted as the loss of efficiency is partially compensated by the greater integration of SYBR® Green molecules in the larger fragments.

Background: Since their first commercialization, the diversity of taxa and the genetic composition of transgene sequences in genetically modified plants (GMOs) are constantly increasing. To date, the detection of GMOs and derived products is commonly performed by PCR-based methods targeting specific DNA sequences introduced into the host genome. Information available regarding the GMOs’ molecular characterization is dispersed and not appropriately organized. For this reason, GMO testing is very challenging and requires more complex screening strategies and decision making schemes, demanding in return the use of efficient bioinformatics tools relying on reliable information. Description: The GMOseek matrix was built as a comprehensive, online open-access tabulated database which provides a reliable, comprehensive and user-friendly overview of 328 GMO events and 247 different genetic elements (status: 18/07/2013). The GMOseek matrix is aiming to facilitate GMO detection from plant origin at different phases of the analysis. It assists in selecting the targets for a screening analysis, interpreting the screening results, checking the occurrence of a screening element in a group of selected GMOs, identifying gaps in the available pool of GMO detection methods, and designing a decision tree. The GMOseek matrix is an independent database with effective functionalities in a format facilitating transferability to other platforms. Data were collected from all available sources and experimentally tested where detection methods and certified reference materials (CRMs) were available. Conclusions: The GMOseek matrix is currently a unique and very valuable tool with reliable information on GMOs from plant origin and their present genetic elements that enables further development of appropriate strategies for GMO detection. It is flexible enough to be further updated with new information and integrated in different applications and platforms.

Next generation sequencing (NGS) is a promising tool for analysing the quality and safety of food and feed products. The detection and identification of genetically modified organisms (GMOs) is complex, as the diversity of transgenic events and types of structural elements introduced in plants continue to increase. In this paper, we show how a strategy that combines enrichment technologies with NGS can be used to detect a large panel of structural elements and partially or completely reconstruct the new sequence inserted into the plant genome in a single analysis, even at low GMO percentages. The strategy of enriching sequences of interest makes the approach applicable even to mixed products, which was not possible before due to insufficient coverage of the different genomes present. This approach is also the first step towards a more complete characterisation of agrifood products in a single analysis.

The persistence and efficacy of biocontrol agents in agricultural fields are crucial for sustainable crop production. In this study, we investigated the persistence of the introduced bacterial strain Pseudomonas sivasensis CF10PS3 in the wheat phyllosphere using a novel qPCR probe protocol. The CF10PS3 strain, known for its in vitro biocontrol properties against wheat pathogens, was applied through foliar spray, and its persistence was monitored over 7 weeks. Our qPCR assays, designed to specifically detect CF10PS3, distinguished it from naturally occurring P. sivasensis strains, providing precise insights into its dynamics in the field. The experimental results indicated that CF10PS3 was already present on the wheat leaves before its application, suggesting its natural adaptation to the foliar environment. Following initial application, a significant increase in CF10PS3 was observed, though subsequent environmental factors such as rain and wind might have caused notable fluctuations in its population. Despite these variations, the introduced strain showed considerable persistence, with population levels significantly higher than those in untreated plots by the end of the study period. This research underscores the importance of understanding bacteria dynamics in the field, highlighting the influence of environmental conditions on their efficacy. The use of specific qPCR probes proved effective in monitoring introduced strains, offering valuable insights for optimizing biocontrol agent application strategies. Our findings contribute to the development of robust biocontrol methods, promoting sustainable agricultural practices and enhancing crop protection.