Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms

AbstractDigital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. This holds true in particular when independent transgene and plant-specific reference gene copy numbers have to be combined to determine the content of GM material in a sample. Droplets which show fluorescent units ranging between those of explicit positive and negative droplets are called ‘rain’. Signals of such droplets can hinder analysis and the correct setting of a threshold. In this manuscript, a computer-based algorithm has been carefully designed to evaluate assay performance and facilitate objective criteria for assay optimization. Optimized assays in return minimize the impact of rain on ddPCR analysis.We developed an Excel based ‘experience matrix’ that reflects the assay parameters of GMO ddPCR tests performed in our laboratory. Parameters considered include singleplex/duplex ddPCR, assay volume, thermal cycler, probe manufacturer, oligonucleotide concentration, annealing/elongation temperature, and a droplet separation evaluation. We additionally propose an objective droplet separation value which is based on both absolute fluorescence signal distance of positive and negative droplet populations and the variation within these droplet populations. The proposed performance classification in the experience matrix can be used for a rating of different assays for the same GMO target, thus enabling employment of the best suited assay parameters. Main optimization parameters include annealing/extension temperature and oligonucleotide concentrations.The droplet separation value allows for easy and reproducible assay performance evaluation. The combination of separation value with the experience matrix simplifies the choice of adequate assay parameters for a given GMO event

Detecting un-authorized genetically modified organisms (GMOs) and derived materials

Genetically modified plants, in the following referred to as genetically modified organisms or GMOs, have been commercially grown for almost two decades. In 2010 approximately 10% of the total global crop acreage was planted with GMOs (James, 2011). More than 30 countries have been growing commercial GMOs, and many more have performed field trials. Although the majority of commercial GMOs both in terms of acreage and specific events belong to the four species: soybean, maize, cotton and rapeseed, there are another 20+ species where GMOs are commercialized or in the pipeline for commercialization. The number of GMOs cultivated in field trials or for commercial production has constantly increased during this time period. So have the number of species, the number of countries involved, the diversity of novel (added) genetic elements and the global trade. All of these factors contribute to the increasing complexity of detecting and correctly identifying GMO derived material. Many jurisdictions, including the European Union (EU), legally distinguish between authorized (and therefore legal) and un-authorized (and therefore illegal) GMOs. Information about the developments, field trials, authorizations, cultivation, trade and observations made in the official GMO control laboratories in different countries around the world is often limited, despite several attempts such as the OECD BioTrack for voluntary dissemination of data. This lack of information inevitably makes it challenging to detect and identify GMOs, especially the un-authorized GMOs. The present paper reviews the state of the art technologies and approaches in light of coverage, practicability, sensitivity and limitations. Emphasis is put on exemplifying practical detection of un-authorized GMOs. Although this paper has a European (EU) bias when examples are given, the contents have global relevance.JRC.I.3-Molecular Biology and Genomic

Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B2 feed additive

Many food and feed additives result from fermentation of genetically modified (GM) microorganisms. For vitamin B2 (riboflavin), GM Bacillus subtilis production strains have been developed and are often used. The presence of neither the GM strain nor its recombinant DNA is allowed for fermentation products placed on the EU market as food or feed additive. A vitamin B2 product (80% feed grade) imported from China was analysed. Viable B. subtilis cells were identified and DNAs of two bacterial isolates (LHL and LGL) were subjected to three whole genome sequencing (WGS) runs with different devices (MiSeq, 454 or HiSeq system). WGS data revealed the integration of a chloramphenicol resistance gene, the deletion of the endogenous riboflavin (rib) operon and presence of four putative plasmids harbouring rib operons. Event- and construct-specific real-time PCR methods for detection of the GM strain and its putative plasmids in food and feed products have been developed.JRC.F.7-Knowledge for Health and Consumer Safet

Bioinformatics DNA sequence datasets.

This data collection shares DNA sequences obtained by the JRC directly from Next Generation Sequencing or from the analysis of public datasets by using Bioinformatics tools. It includes DNA sequences related to Genetically Modified Organisms, like plants and micro-organisms.JRC.F.7-Knowledge for Health and Consumer Safet

Guidance document from the European Network of GMO Laboratories (ENGL): Overview on the detection, interpretation and reporting on the presence of unauthorised genetically modified materials

Unauthorised GMOs (UGM) and derived materials are not uncommon in products found on the European market. At present there is zero tolerance for UGM in the EU. In most documented cases, the UGM concentration relative to the product in which the UGM material is found, was low. Low level presence will always represent a challenge to analytically based detection, in particular if the UGM is obscured by other GM material. Analytical ad hoc implementation of the zero tolerance for particular UGMs in feed is described in Regulation (EU) No 619/2011. In Europe, GM detection is predominantly achieved with polymerase chain reaction (PCR) derived methods targeting the transgenic construct and insertion site DNA sequences. The increase in number and divergence of GMOs developed and commercialised has gradually forced the GM detection laboratories to rationalise their analytical work, and most laboratories now apply initial PCR based screenings followed by (when appropriate) more specific PCR based identification and quantification. The detection of any GM is dependent on availability of suitable detection method(s) and control materials to verify the performance of the method(s). Other information, e.g. describing the novel trait, introduced genetic elements, etc. may also facilitate detection, verification and identification of the GM. For UGM, this is a major challenge, and the GMOs are therefore classified into four knowledge groups in the present document. This classification may facilitate stakeholder communication and decision making in analytical laboratories. A decision tree is presented, summarising the recommended principles of GM and UGM detection. Notably, the state-of-the-art of GMO analysis is not static, and it is expected that the guidelines and recommendations presented in this document will have to be modified on a regular basis. Finally, the document highlights a number of R&D priorities and points out the need for reinforced information sharing at the global level

Overview on the detection, interpretation and reporting on the presence of unauthorised genetically modified materials : Guidance document from the European Network of GMO Laboratories (ENGL)

Unauthorised GMOs (UGM) and derived materials are not uncommon in products found on the European market. At present there is zero tolerance for UGM in the EU. In most documented cases, the UGM concentration relative to the product in which the UGM material is found, was low. Low level presence will always represent a challenge to analytically based detection, in particular if the UGM is obscured by other GM material. Analytical ad hoc implementation of the zero tolerance for particular UGMs in feed is described in Regulation (EU) No 619/2011. In Europe, GM detection is predominantly achieved with polymerase chain reaction (PCR) derived methods targeting the transgenic construct and insertion site DNA sequences. The increase in number and divergence of GMOs developed and commercialised has gradually forced the GM detection laboratories to rationalise their analytical work, and most laboratories now apply initial PCR based screenings followed by (when appropriate) more specific PCR based identification and quantification. The detection of any GM is dependent on availability of suitable detection method(s) and control materials to verify the performance of the method(s). Other information, e.g. describing the novel trait, introduced genetic elements, etc. may also facilitate detection, verification and identification of the GM. For UGM, this is a major challenge, and the GMOs are therefore classified into four knowledge groups in the present document. This classification may facilitate stakeholder communication and decision making in analytical laboratories. A decision tree is presented, summarising the recommended principles of GM and UGM detection. Notably, the state-of-the-art of GMO analysis is not static, and it is expected that the guidelines and recommendations presented in this document will have to be modified on a regular basis. Finally, the document highlights a number of R&D priorities and points out the need for reinforced information sharing at the global level.JRC.I.3-Molecular Biology and Genomic

Incidence and prognostic factors related to major adverse cerebrovascular events in patients with complex aortic diseases treated by the chimney technique

Objective: Endovascular aneurysm repair (EVAR) with the chimney technique (ch-EVAR) has been used for the treatment of aortic aneurysms as an alternative approach to fenestrated endografting or open repair. Nonetheless, the need for an upper extremity arterial access may contribute to a higher risk for periprocedural cerebrovascular events. This study reports on the perioperative cerebral and major adverse cardiac and cerebrovascular events (MACCE) after ch-EVAR. Methods: The PERICLES registry (PERformance of the chImney technique for the treatment of Complex aortic pathoLogiES) is an international, retrospective multicenter study evaluating the performance of ch-EVAR for the treatment of complex aortic pathologies. For the purpose of the current analysis, 425 patients treated by ch-EVAR between 2008 and 2014 were included. The primary outcome of this analysis was the incidence of procedure related cerebrovascular events defined as transient ischemic attack or stroke. The secondary end point was in-hospital MACCE, including acute coronary syndrome, stroke, and death of any cause. Results: The incidence of clinical relevant cerebrovascular events was 1.9% (8/425). A postoperative transient ischemic attack was observed in four patients (0.95%) and a stroke in additional four (0.95%). Three patients died during the hospital stay secondary to sequelae from postoperative stroke. A prior history of stroke/transient ischemic attack, atrial fibrillation, previous carotid revascularization, or known carotid artery disease did not significantly increase the risk for adverse neurologic events. The overall MACCE rate amounted to 8.5% (36/425). Logistic regression analysis revealed that the use of bilateral upper extremity access (odds ratio [OR], 2.79; 95% confidence interval [CI], 1.04-7.45]), aneurysm rupture (OR, 5.33; 95% CI, 1.74-16.33), and a prolonged operation time (>290 minutes; OR, 1.005; 95% CI, 1.001-1.008) were associated with a significantly increased risk for MACCE. Conclusions: This analysis demonstrates that ch-EVAR is associated with a relatively low rate of cerebrovascular events. However, a postoperative stroke is associated with increased mortality. Ruptured aneurysms, bilateral upper extremity access as in case of multiple chimney graft placement, and longer operative times were identified as independent risk factors for MACCE