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Technical Tools and Guidance for the Detection and Identification of LMOs

Overview of available databases of methods, and screening matrices for the detection of living modified organisms

By: Bjørn Spilsberg and Lutz Grohmann

Databases of methods for the detection of living modified organisms

Databases containing accurate and reliable information on methods, reference materials and DNA sequences, if available, are an important tool to enable countries to effectively detect and identify Living Modified Organisms (LMOs). To be useful to the public at large, such databases must, at a minimum, be available online and open to the public, contain accurate and up-to-date information, and have user-friendly mechanisms for searching and retrieving information. The scope of the database may vary in the type of methods (e.g. DNA and/or protein; validated or not), type of LMOs (e.g. crops, vaccines, etc) or geographic area (e.g. local or global).

Below is information on databases that meet these criteria. They include databases of detection methods and reference materials.

European Union Database of Reference Methods for GMO Analysis

This database was developed by the Joint Research Centre’s European Union Reference Laboratory for Genetically Modified Food and Feed (EURL-GMFF). It is maintained by the EURL-GMFF in collaboration with the European Network of GMO Laboratories (ENGL). This database contains fully validated detection methods for LMOs using, mostly, real-time PCR methods as well as some gel-based PCR methods. The collection includes methods that are event-specific, construct-specific and element-specific methods for screening as well as plant taxon-specific methods for species identification. For each method a complete protocol with the information needed to conduct the test and all the validation data are provided.

Event-specific detection methods validated by the EURL-GMFF can also be found by searching the "Status of Dossiers" list on the EURL-GMFF website.

GMO Detection Method Database (GMDD)

GMO Detection Method Database (GMDD) was developed and is maintained by the GMO Detection Laboratory at Shanghai Jiao Tong University in China. It contains information on event-specific detection methods for many LMOs, as well as real-time PCR and gel-based PCR methods that are element-specific, gene-specific or reference gene specific. Some protein-based methods are also included. Not all the methods in the database are validated through collaborative trial studies, however, if available, information on the validation status of a method is provided. The primer and probe sequences are provided with citations to the relevant publications. The database also includes a large collection of publicly available LMO sequence information. In addition, links to GenBank and other publications containing information on the sequence of the inserts is provided. Finally, there is also information on relevant certified reference materials (CRM), if available.

CropLife International Detection Methods Database

This database is maintained by CropLife International, a global federation of the plant biotechnology industry, and contains both DNA- and protein-based methods. It currently contains approximately 40 methods related to about 27 LMOs. The detection methods available have been developed and validated by the technology providers for their own proprietary technologies and products. Not-for-profit laboratories have free access to the methods, whereas laboratories that undertake “fee-for-service” testing must request a license to access the methods.

Screening matrices for the detection of living modified organisms

The number of commercially available LMOs is huge and is growing at the global scale. It is therefore not efficient to only employ event-specific methods when testing for LMO presence. Fortunately, there is a considerable body of knowledge in the scientific literature that contains valuable information on strategies that have been developed for the identification of LMOs. The most cost efficient alternative strategy to detect the potential presence of an LMO in a sample is to use a screening approach which employs the combination of element-specific detection methods selected on the basis of information on presence or absence of the target in a list of LMOs.

There are two different types of commonly used matrices as described in the recently drafted European Technical Specification for the application of PCR-based screening strategies using the so called “matrix approach”. According to this document, the first type of screening matrix is the “GMO method matrix”, is defined as a relational presentation, for example a table, of symbols or numbers, where the genetic elements and genetic constructs that are detected by a defined PCR method and the corresponding LMOs are tabulated. The symbols (“+” or “-”, or numbers) indicate whether or not the target sequence is detectable by the specified method in a given LMO.

The second type of screening matrix, the “GMO target matrix”, tabulates only information about the presence of genetic elements or genetic constructs in LMOs. The entered symbols or numbers indicate the presence or absence of the target sequence and copy number, if available, in a given LMO event. In contrast to “GMO method matrices”, “GMO target matrices” are independent from a particular detection method.

German laboratory network Screening Table

The German laboratory network has developed a “GMO method matrix” (also known as the Waiblinger Table), which is based on five methods targeting specific genetic elements and constructs that are most frequently present in commercialized LM crops. It comprises a set of real-time PCR methods to detect:
  1. the Cauliflower Mosaic Virus 35S promoter (P-35S);
  2. the nos terminator derived from Agrobacterium tumefaciens (T-nos);
  3. the ctp2-cp4epsps junction of the chloroplast-transitpeptide (CTP2) from Arabidopsis thaliana and the epsps gene from A. tumefaciens strain CP4 (epsps);
  4. the bar gene from Streptomyces hygroscopicus; and
  5. a sequence from the P35S-pat junction of the CaMV P-35S promoter and the synthetic pat gene3.

All five methods have been fully validated and are included in the EURL-GMFF method database. Furthermore a table listing the accessibility of publicly available reference materials and their sources is provided. The Screening Table is maintained by the German NRL and the Excel spreadsheet with implemented filter-functionalities and a list of available LMO reference materials can be downloaded from the internet.

GMOseek matrix

The GMOseek matrix is a ‘GMO target matrix’ and provides a comprehensive and user-friendly overview of 273 genetic elements and their occurrence in 328 LMOs5. The GMOseek matrix is freely available online as an Excel spreadsheet. Filtering functions allow users to search for events that fit into a defined pattern of genetic elements based on their absence or presence in an LMO. It also helps users in identifying genetic elements that could be targeted during the screening phase of LMO analysis.

JRC GMO-Matrix

The JRC GMO-Matrix is a “GMO method matrix”. It takes advantage of the DNA sequence information compiled in the JRC’s Central Core DNA Sequence Information System (CCSIS). The JRC receives DNA sequence information on the insertions in LMOs from plant biotechnology companies, as part of their legal obligations in the EU. In addition they extract sequence information from nucleotide or patent sequence databases, as well as the primer and probe sequences of the detection methods compiled in the EURL-GMFF reference method database.

The user selects a set of plant species and/or LMOs and a set of detection methods and after in silico simulations of PCR amplification using bioinformatics tools, the results are displayed in a table with predictions of possible amplification(s).

GMOfinder

The GMOfinder is a compilation of data to construct a combination of both, a ‘GMO target matrix’ and a ‘GMO method matrix’. It is based on an MS Access database and has integrated algorithms that facilitate the interpretation of the results of screening analyses. The tabular matrix provides information on selected genetic elements originating from the literature, LMO notifications and other sources. This information is integrated in a tabular format for 15 real-time PCR methods partly targeting the same genetic element but having different ranges of specificity. The recording of the sources of information facilitates a careful evaluation of the screening results and the tracing back of possible errors in the conclusions of the screening analysis. The GMOfinder is available free of charge upon requests addressed to the authors.

Combinatory qPCR SYBR® Green screening

Combinatory qPCR SYBR® Green screening (CoSYPS) is a “GMO method matrix” based on the SYBR® Green qPCR analysis method for detecting the presence of the following genetic elements in LMOs: the Cauliflower Mosaic Virus 35S promoter and terminator, the nos promoter and terminator derived from Agrobacterium tumefaciens, the Figwort Mosaic Virus promoter, the rice actin promoter, the nptII gene from Escherichia coli, the epsps gene from Agrobacterium tumefaciens CP4, the epsps gene from Zea mays, the pat gene from Streptomyces viridochromogenes, the bar gene from Streptomyces hygroscopicus, the barnase gene from Bacillus amyloliquefaciens and several cry genes (cry1Ab, cryAc, cryF, cry3Bb) from Bacillus thuringiensis. In addition, a set of plant taxon-specific method is included in the CoSYPS testing platform.

The analytical results obtained with the CoSYPS matrix are interpreted and evaluated in combination with a “prime number”-based algorithm, by which the nature of the subsets of corresponding LMOs in a sample can be determined.

GMO Checker

This screening application was developed as a real-time PCR array and is a ‘GMO method matrix’. The platform can be used for the comprehensive and semi-quantitative detection of LM crops. It is a combination of 14 event-specific and 10 element specific methods. The specificity and sensitivity of the PCR assays were evaluated experimentally and are tabulated in the publication. An Excel spreadsheet application for the evaluation of analytical results concerning the presence of LM crops has been developed and can be downloaded from the internet.

Other relevant databases

Biosafety Clearing-House

The Biosafety Clearing-House (BCH) central portal contains an LMO Registry with detailed descriptions for each LMO, including a unique identifier, if available, and detailed information on the transformation method, modified genetic elements, and vector as well as links to other registries in the BCH such as for risk assessments and countries’ decisions. Each entry in the LMO registry also contains links to relevant detection methods for many of the commercialized LMOs.

The BCH also contains two other registries which are closely related to the LMO registry: the Organism Registry and the Genetic Element Registry. The Organism Registry includes information on the donor organisms and the recipient or parental organisms for the registered LMOs.

The Genetic Element Registry contains records of the genes and other genetic elements that were modified in the LMOs. For each entry there is a brief description of the element and links to associated LMOs. Due to the confidential nature of the information, actual sequence information is available for only a few of the genetic elements.

BioTrack Product Database

The Biotrack Product database is maintained by the Organisation for Economic Co-operation and Development (OECD). It compiles a list of Unique Identifiers (UIs) for LM plants that have been approved for commercial application in at least one country, in terms of food, feed or environmental safety. UIs are codes of a fixed length of 9 alphanumeric digits specific for a single transformation event and are intended to be used as "keys" to access and share information on a particular LMO.

BIOTradeStatus

The BIOTradeStatus database is maintained by the Biotechnology Industry Organization and contains information on approvals, commercialization and seed sale of common commercially available LMOs.

CERA LM Crop Database

The Center for Environmental Risk Assessment (CERA), established by the food-industry funded, non-profit International Life Science Institute Research Foundation (ILSI), also maintains an LM crop database which includes not only plants produced using recombinant DNA technologies (e.g., genetically engineered or transgenic plants), but also plants with novel traits that may have been produced using more traditional methods, such as accelerated mutagenesis or plant breeding. The database provides information on the genetic elements construct, vector as well as the LMO characteristics (traits, common use etc.), risk assessments and regulatory decisions.

GMOtrack

This program generates cost-effective testing strategies for traceability of LMOs and computes the optimal set of screening assays for a two-phase testing strategy.


Resources

In this paper, it is shown that the combination of five DNA target sequences can be used as a universal screening approach for at least 81 GM plant events authorised or unauthorised for placing on the market and described in publicly available databases. Anal.Bioanal.Chem. 2010, 396, 2065-72.
The Biotrack Product database compiles a list of Unique Identifiers (UIs) for LM plants that have been approved for commercial application in at least one country.
The BIOTradeStatus database is maintained by the Biotechnology Industry Organization and contains information on approvals, commercialization and seed sale of common commercially available LMOs.
CERA's LM crop database.
“Combinatory qPCR SYBR®Green screening” (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. Anal Bbioanal Chem 2010, 396, 2113-23.
A global federation of private plant biotechnology developers, and contains both DNA- and protein-based methods
Maintained by the Joint Research Center of the European Commission
A table listing the current status of dossiers within the EU-RL GMFF validation process carried out within the frame of the Regulation (EC) No 1829/2003.
In this database, users can obtain the sequences of exogenous integration, which will facilitate PCR primers and probes design. Also the information on endogenous genes, certified reference materials, reference molecules, and the validation status of developed methods is included in this database. BMC Bioinformatics 2008, 9, 260.
This screening application was developed as a real-time PCR array and is a ‘GMO method matrix’. The platform can be used for the comprehensive and semi-quantitative detection of LM crops.
Maintained by the GMO Detection Laboratory at Shanghai Jiao Tong University.
GMOfinder is a database for collection and interpretation of information related to the screening for genetically modified organisms (GMOs). Food Anal. Methods, 2012, 5, 1368–1376
The GMOMETHODS database currently contains 118 different PCR methods allowing identification of 51 single GM events and 18 taxon-specific genes in a sample. J. AOAC Int. 2012, 95, 1713-9.
This program generates cost-effective testing strategies for traceability of LMOs and computes the optimal set of screening assays for a two-phase testing strategy.
A novel platform that links the information of two unique databases built and maintained by the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) at the Joint Research Centre (JRC) of the European Commission, one containing the sequence information of known GM-events and the other validated PCR-based detection and identification methods. The new platform compiles in silico determinations of the detection of a wide range of GMOs by the available detection methods using existing scripts that simulate PCR amplification and, when present, probe binding.
A study of the application of an informative detailed 24-element screening and subsequent identification strategy was applied in 50 animal feed samples. Agric Food Chem. 2013, 61, 9097-109
A novel type of real-time polymerase chain reaction (PCR) array with TaqMan chemistry as a platform for the comprehensive and semiquantitative detection of genetically modified (GM) crops. J Agric Food Chem., 2009, 57, 26-37
A table listing the accessibility of publicly available reference materials and their sources is presented with this method matrix.
The program is designed to assist in selecting the optimal combination of screening methods and interpreting the data from screening and event-specific testing.
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. BMC Bioinformatics 2013, 14, 256
A GMO method matrix which is based on 5 methods targeting specific genetic elements and constructs that are most frequently present in commercialized LM crops.