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Record information and status
Record ID
Date of creation
2019-07-31 20:02 UTC (austein.mcloughlin@cbd.int)
Date of publication
2019-07-31 20:02 UTC (austein.mcloughlin@cbd.int)

General Information
Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179
Patrick Guertler, Lutz Grohmann, Heike Naumann, Melanie Pavlovic, Ulrich Busch
  • English
Publication date
Summary, abstract or table of contents

Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand,
cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on patent sequence information, event-specific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed. Newly developed plasmids were used as reference material for assay optimization and in-house validation. Plasmid standards were quantified using digital droplet PCR and LOD95%, PCR efficiency, robustness and specificity of the assays were determined using real-time PCR. A LOD95% of 10 copies per PCR reaction was observed and PCR efficiencies of 95-97 % were achieved. Different real-time PCR instruments and PCR conditions were applied to test for robustness of the assays using DNA at a concentration of 30 copies per μL for each gm alfalfa event. All replicates were positive independent of the instrument or the PCR condition. DNA from certified reference material of different genetically modified crops as well as reference materials of the three events was used to experimentally test for specificity. No unspecific amplification signal was observed for any of the assays. Validation results were in line with the "Minimum Performance Requirements for Analytical Methods of GMO Testing" of the European Network of GMO Laboratories. Furthermore, an inter-laboratory comparison study was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters.
Thematic areas
Information on Organisms or LMOs
LMO(s) identification
MON-ØØ163-7 - Roundup Ready™ alfalfa
Resistance to herbicides - Glyphosate
MON-ØØ1Ø1-8 - Roundup Ready™ alfalfa
Resistance to herbicides - Glyphosate
MON-ØØ179-5 - Reduced Lignin Alfalfa
Changes in quality and/or metabolite content - Lignin Resistance to antibiotics - Kanamycin
Additional Information
Type of resource
  • Article (journal / magazine / newspaper)
DOI: 10.1016/j.bdq.2018.12.001
Publisher and its location
Biomolecular Detection and Quantification
PDF file: 7 pages (900 KB)
Article from a journal
Keywords and any other relevant information
Detection, Identification, Alfalfa, PCR, qPCR