Yves Bertheau

A microarray-based detection system for genetically modified (GM) food ingredients

A multiplex DNA microarray chip was developed for simultaneous identification of nine genetically modified organisms (GMOs), five plant species and three GMO screening elements, i.e. the 35S promoter, the nos terminator and the nptII gene. The chips also include several controls, such as that for the possible presence of CaMV. The on-chip detection was performed directly with PCR amplified products. Particular emphasis was placed on the reduction of the number of PCR reactions required and on the number of primers present per amplification tube. The targets were biotin labelled and the arrays were detected using a colorimetric methodology. Specificity was provided by specific capture probes designed for each GMO and for the common screening elements. The sensitivity of the assay was tested by experiments carried out in five different laboratories. The limit of detection was lower than 0.3% GMO for all tests and in general around 0.1% for most GMOs. The chip detection system complies with the requirements of current EU regulations and other countries where thresholds are established for the labelling of GMO.

Detection of depolymerase isoenzymes after electrophoresis or electrofocusing, or in titration curves☆

The cup-plate technique makes it possible to detect enzyme activities after diffusion into buffered, substrate-containing agar gels. This technique has been used after nondenaturing blotting transfer in order to detect depolymerizing enzyme activities once analytical protein separation (e.g., by electrophoresis, electrofocusing, or titration curves) has been completed. This rapid and very sensitive method was successfully applied to the enzymes polygalacturonate lyase, polygalacturonate hydrolase, endoglucanase, and xylan hydrolase. Other possible applications are presented.

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.

Molecular cloning of Erwinia chrysanthemi pectinase and cellulase structural genes

Erwinia chrysanthemi 3937 secretes four major pectate lyase isoenzymes (PL, EC 4.2.2.2) and one endocellulase (Cx, EC 3.2.1.4). A genomic library of this strain was constructed in the Lambda L47‐1 vector, and screened for the presence of PL and Cx on pectate and caboxymethylcellulose agar. Among the seven Cx‐positive phage clones, three were shown to encode an enzyme of the same mol. wt. as the one found in the culture supernatant of strain 3937. The 34 PL‐positive phage clones were analyzed by electrofocusing and could, according to the PL they produced, be arranged in five classes. Phages from three classes produced three different single PL, named PLb, c and d. No common fragment was evidenced between the inserts of the phages of these three classes. This demonstrated that, in strain 3937, PLb, C, and d were encoded by three different genes called pelB, C, and D. Furthermore, our results suggest the existence of two additional genes encoding PLa and e. In addition, a pectin methylesterase gene was found closely linked to pelD.

Gene stacking in transgenic plants: towards compliance between definitions, terminology, and detection within the EU regulatory framework.

The combination or stacking of different traits or genes in plants is rapidly gaining popularity in biotech crop production. Here we review the existing terminology regarding gene stacking in plants, and its implications in relation to genetics, biosafety, detectability and European regulations. Different methods of production of stacked gene traits, as well as the status of their cultivation and approval, are reviewed. Related to the different techniques of transformation and production, including classical breeding, and to differences in global authorization and commercialization practices, there are many types, definitions, and perceptions of stacking. These include: (1) stacking of traits and (2) stacking of events, which are the most widely accepted perceptions of stacking, and (3) stacking of genes, which from the analytical and traceability point of view may be a more appropriate perception. These differences in perceptions and definitions are discussed, as are their implications for analytical detection and regulatory compliance according to (in particular) European Union (EU) regulations. A comprehensive terminology regarding gene stacking with regulatory relevance is proposed. The haploid genome equivalent is proposed as the prevailing unit of measurement at all stages throughout the chain, in order to ensure that terminology and definitions of gene stacks are adapted to analytical detection, traceability, and compliance with EU regulations.

Detection of nonauthorized genetically modified organisms using differential quantitative polymerase chain reaction: application to 35S in maize

Detection of nonauthorized genetically modified organisms (GMOs) has always presented an analytical challenge because the complete sequence data needed to detect them are generally unavailable although sequence similarity to known GMOs can be expected. A new approach, differential quantitative polymerase chain reaction (PCR), for detection of nonauthorized GMOs is presented here. This method is based on the presence of several common elements (e.g., promoter, genes of interest) in different GMOs. A statistical model was developed to study the difference between the number of molecules of such a common sequence and the number of molecules identifying the approved GMO (as determined by border-fragment-based PCR) and the donor organism of the common sequence. When this difference differs statistically from zero, the presence of a nonauthorized GMO can be inferred. The interest and scope of such an approach were tested on a case study of different proportions of genetically modified maize events, with the P35S promoter as the Cauliflower Mosaic Virus common sequence. The presence of a nonauthorized GMO was successfully detected in the mixtures analyzed and in the presence of (donor organism of P35S promoter). This method could be easily transposed to other common GMO sequences and other species and is applicable to other detection areas such as microbiology.

Characterization and overexpression of the pem gene encoding pectin methylesterase of Erwinia chrysanthemi strain 3937

The pem gene encoding the pectin methylesterase (PME) of Erwinia chrysanthemi strain 3937 was subcloned and its nucleotide sequence determined. The gene contains an open reading frame of 1098 bp and codes for a protein of 366 amino acids (aa). The mature 37-kDa form of the protein is 342 aa long and has a calculated isoelectric point of 9.64. A plasmid was constructed to overproduce PME: a DNA fragment carrying pem was amplified by the polymerase chain reaction and cloned downstream from the pL promoter of the lambda phage, in a high-copy-number plasmid. In an Escherichia coli strain transformed with this plasmid, an increase in PME production of more than 60-fold was obtained, compared with the wild-type Er. chrysanthemi strain. PME represents about 5% of the total protein content of the cells. Comparison of this PME sequence with six PMEs from prokaryotic or eukaryotic organisms showed six highly conserved segments whose possible role in enzyme activity are discussed.

Evaluation of a PCR kit for the detection of Erwinia carotovora subsp. atroseptica on potato tubers

SummaryA PCR-based kit, ProbeliaTM, for the detection ofErwinia carotovora subsp.atroseptica (Eca) on potatoes was evaluated at five laboratories in four countries. The kit is based on DNA-specific PCR amplification followed by detection of amplicons by hybridization to a peroxidase-labelled DNA probe in a microplate. Specificity of the PCR primers for Eca, regardless of serogroups, was confirmed by testing against 246 bacterial, fungal and plant species. Detection limits of the assay varied little between six Eca strains in pure cultures (1.3×102 to 1.5×103 cells ml−1). When Eca-free tuber peel extract from four cultivars was inoculated with known numbers of 15 Eca strains, detection limits were more variable (1.0×101 to 6.2×103 cells ml−1 peel extract), attributed probably to inconsistency in the recovery of DNA during extraction. When the PCR assay was compared with three current commercial Eca detection methods, using naturally contaminated tubers, results matched most closely those from viable counts on a selective medium, the most sensitive method (88%), followed by enrichment ELISA (72%) and last ELISA (30%), the least sensitive method.

Development of a Real-Time PCR Method for the Differential Detection and Quantification of Four Solanaceae in GMO Analysis: Potato (Solanum Tuberosum), Tomato (Solanum Lycopersicum), Eggplant (Solanum Melongena), and Pepper (Capsicum Annuum)

The labeling of products containing genetically modified organisms (GMO) is linked to their quantification since a threshold for the presence of fortuitous GMOs in food has been established. This threshold is calculated from a combination of two absolute quantification values: one for the specific GMO target and the second for an endogenous reference gene specific to the taxon. Thus, the development of reliable methods to quantify GMOs using endogenous reference genes in complex matrixes such as food and feed is needed. Plant identification can be difficult in the case of closely related taxa, which moreover are subject to introgression events. Based on the homology of beta-fructosidase sequences obtained from public databases, two couples of consensus primers were designed for the detection, quantification, and differentiation of four Solanaceae: potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena). Sequence variability was studied first using lines and cultivars (intraspecies sequence variability), then using taxa involved in gene introgressions, and finally, using taxonomically close taxa (interspecies sequence variability). This study allowed us to design four highly specific TaqMan-MGB probes. A duplex real time PCR assay was developed for simultaneous quantification of tomato and potato. For eggplant and pepper, only simplex real time PCR tests were developed. The results demonstrated the high specificity and sensitivity of the assays. We therefore conclude that beta-fructosidase can be used as an endogenous reference gene for GMO analysis.

Evaluation of phenotypic and molecular typing techniques for determining diversity in Erwinia carotovora subsp. atroseptica

A number of phenotypic and molecular fingerprinting techniques, including physiological profiling (Biolog), restriction fragment length polymorphism (RFLP), enterobacterial repetitive intergenic consensus (ERIC) and a phage typing system, were evaluated for their ability to differentiate between 60 strains of Erwinia carotovora ssp. atroseptica (Eca) from eight west European countries. These techniques were compared with other fingerprinting techniques, random amplified polymorphic DNA (RAPD) and Ouchterlony double diffusion (ODD), previously used to type this pathogen. Where possible, data were represented as dendrograms and groups/subgroups of strains identified. Simpsons index of diversity (Simpsons D) was used to compare groupings obtained with the different techniques which, with the exception of Biolog, gave values of 0·46 (RFLP), 0·39 (ERIC), 0·83 (phage typng), 0·82 (RAPD) and 0·26 (ODD). Of the techniques tested, phage typing showed the highest level of diversity within Eca, and this technique will now form the basis of studies into the epidemiology of blackleg disease.