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Evaluation of the reliability of maize reference assays for GMO quantification

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Abstract

A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize.

Comparison of performance of reference assays in worldwide collection of varieties. Each scatter plot was constructed by plotting the copy numbers determined with one reference assay (x-axis) against the copy numbers determined with another reference assay (y-axis) after the genewise normalisation of copy number data. The red line in each scatter plot denotes a perfect correlation (r = 1) of copy numbers between the two assays. The individual correlation coefficients between the assays are also shown in the squares diagonally opposite to individual plots

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Abbreviations

Adh1 :

Alcohol dehydrogenase 1 gene

BLAST:

Basic Local Alignment Search Tool

Bp:

Base pairs

CE:

Capillary electrophoresis

CRL:

Community Reference Laboratory

CRMs:

Certified reference materials

CSCE:

Conformation sensitive capillary electrophoresis

CSIC:

Consejo Superior de Investigaciones Cientificas

CV:

Coefficient of variation

DNA:

Deoxyribonucleic acid

dNTP:

Deoxynucleotide triphosphate

EC:

European Commission

EU:

European Union

GM:

Genetically modified

GMO:

Genetically modified organism

Hmg :

High mobility group protein gene

ILVO:

Institute for Agricultural and Fisheries Research

Indel:

Insertion/deletion

IRMM:

Institute for Reference Materials and Measurements

Ivr :

Invertase gene

JRC:

Joint Research Centre

NIB:

National Institute of Biology

PCR:

Polymerase chain reaction

RA:

Reference assay

SNP:

Single nucleotide polymorphism

SSIIb :

Zea mays starch synthetase type B gene

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Acknowledgements

This work was financially supported by the European Commission through the Sixth Framework Programme, integrated project Co-Extra (http://www.coextra.eu), contract FOOD-2005-CT-007158 and by the Slovenian Research Agency Programme Plant biotechnology and systems biology P4-0165. Maria Pla (CSIC, Spain) and Zoran Čergan (Agricultural Institute of Slovenia) are acknowledged for providing seed and DNA material from maize varieties. We are very thankful to Cindy Merckaert and Kato Platteau for the excellent experimental work.

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Authors and Affiliations

  1. Technology and Food Sciences, Institute for Agricultural and Fisheries Research (ILVO), Burg. Van Gansberghelaan 115, 9820, Merelbeke, Belgium

    Nina Papazova, Marc De Loose & Isabel Taverniers

  2. GEVES Domaine du Magneraud, Laboratoire BioGEVES, B.P. 52, 17700, Surgères, France

    David Zhang & Stephane Fouilloux

  3. Department of Biotechnology and Systems Biology, National Institute of Biology (NIB), Vecna pot 111, 1000, Ljubljana, Slovenia

    Kristina Gruden, Jana Vojvoda, Meti Buh Gašparič & Andrej Blejec

  4. GMO Detection Laboratory, SJTU-Bor Luh Food Safety Center, School of life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China

    Litao Yang

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  1. Nina Papazova
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  2. David Zhang
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  3. Kristina Gruden
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  9. Marc De Loose
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  10. Isabel Taverniers
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Correspondence to Isabel Taverniers.

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Nina Papazova and David Zhang contributed equally to this paper.

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Papazova, N., Zhang, D., Gruden, K. et al. Evaluation of the reliability of maize reference assays for GMO quantification. Anal Bioanal Chem 396, 2189–2201 (2010). https://doi.org/10.1007/s00216-009-3386-4

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