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Parallelised real-time PCR for identification of maize GMO events

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Abstract

Identification of specific material derived from genetically modified organisms (GMO) present in food, feed or seed samples screened positive for the presence of genetic modification(s) is mandatory for the official food and feed control in the European Union. Since the introduction of regulation (EC) No 1829/2003 in 2004, the number of maize GMO events either approved in the EU or with a pending application grew constantly. By the sheer multitude of events and crossed events (stacks), maize poses a special challenge on official food and feed control. We developed a modular qualitative detection system for the parallel identification of maize GMO events to cope with the increasing number of GMO potentially present in routine samples. This system is based on validated real-time PCR assays in a microtitre plate format grouped modularly by crop species. The maize module identifies in parallel, i.e. simultaneously, 15 maize events and RoundupReady soy in a single analytical run of approximately 2 h. Maize modules can be conveniently prepared in advance and stored at −20 °C until use. Ready-to-use reference DNA mixtures serve as positive controls. The modular approach is flexible as it allows easy change or addition of individual detection reactions, if necessary, e.g. when new validated methods become available. 23 food, 14 feed and 8 seed samples were successfully analysed with the maize module. The parallel detection of nine different GMO maize and soy events in single routine samples demonstrated the usefulness of the parallelised modular approach for routine GMO analysis.

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Abbreviations

CTAB:

Cetyltrimethylammonium bromide

EFSA:

European Food Safety Authority

ENGL:

European Network of GMO Laboratories

EURL-GMFF:

European Reference Laboratory for GM Food and Feed

FAM:

Fluorescein

gDNA:

Genomic DNA

gm:

Genetically modified

GMO:

Genetically modified organism

HSD:

Herring sperm DNA

MDA:

Multiple displacement amplification

MGBNFQ:

Minor groove binder/non-fluorescent quencher

PCR:

Polymerase chain reaction

RRS:

RoundupReady soy (event 40-3-2)

TAMRA:

Tetramethylrhodamin

UNG:

Uracil-DNA-glycosylase

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Acknowledgments

We thank Krimhilde Posthoff, Claudia Bujotzek, Ulrike Mulats, Melina Mehmedovic, Roswitha Dorfner and Manuela Hillen for excellent technical assistance. We would like to acknowledge Ottmar Goerlich for providing plant material and DNA preparations. The presented work was funded by the Bavarian State Ministry of the Environment and Public Health.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Bavarian Health and Food Safety Authority (LGL), Veterinärstr. 2, 85764, Oberschleißheim, Germany

    Lars Gerdes, Ulrich Busch & Sven Pecoraro

Authors
  1. Lars Gerdes
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  2. Ulrich Busch
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  3. Sven Pecoraro
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Corresponding author

Correspondence to Sven Pecoraro.

Electronic supplementary material

Below is the link to the electronic supplementary material.

217_2011_1634_MOESM1_ESM.xls

Online Resource 1 Positive control mixture for the maize module Excel spreadsheet used for calculation of positive control mixture for the maize module. Instructions for use are given on the spreadsheet and in the included commentaries. (XLS 32 kb)

217_2011_1634_MOESM2_ESM.xls

Online Resource 2 Assays used in the maize module Assays for all GMO events used in the maize module were listed with unique identifier (UI) of the event, source of reference material (Ref. Material), method, concentrations (/nM) and sequences of forward primer (fw), reverse primer (rv), and probe (pr) with fluorescence label. (XLS 19 kb)

217_2011_1634_MOESM3_ESM.ppt

Online Resource 3 Assay performances after storage Cq values for the assays of three lots of maize modules are depicted after different periods of storage at –20 °C. Times refer to days (d) after preparation. The two crosses in lot ‘100608’ (NK603) and lot ‘100317’ (LY038) depict failed amplifications due to errors in preparation of the positive control mixtures. Most of the variations are restricted to a range of less than two Cq units. (PPT 229 kb)

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Gerdes, L., Busch, U. & Pecoraro, S. Parallelised real-time PCR for identification of maize GMO events. Eur Food Res Technol 234, 315–322 (2012). https://doi.org/10.1007/s00217-011-1634-2

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