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Evaluation of Adh1 alleles and transgenic soybean seeds using Scorpion PCR and HRM analysis

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Abstract

The identification of both approved and non-approved genetically modified organisms (GMOs) is an integral part of GMO biosafety legislation in many countries. One aspect that may affect PCR-based detection of a GMO lies within the analysis of its genetic stability, as sequence alterations or DNA instabilities may impede quantification by PCR. Genetic stability can be analyzed using various methods, yet many of these methods have distinct disadvantages, including low sensitivity. In this study, high resolution melting (HRM) analysis and real-time PCR with Scorpion primers were used as a method to analyze the 3′ end of RR soybeans (RR 40-3-2) in a large number of samples (n = 1,034). No evidence for the occurrence of mutation events was found, implying that the nucleotide sequence of this region is unlikely to be unstable and is well suited as a target for the quantification of RR soybeans. Additionally, and as a preparative work for an optimization of the method, a 174 bp region of the first intron of the Adh1 gene was analyzed in several varieties of maize with different GMO events using the same approach. The results show that 2 alleles are present. In further experiments, the different alleles were cloned into plasmids to generate homozygous plasmids from heterozygous templates in order to generate for a more precise analysis. The overall methodological aim of these studies was to compare HRM analysis with Scorpion primer PCR. Both methods were capable of differentiating between the 2 homozygous and heterozygous alleles. For a better discrimination, however, we conclude that it is most reliable to consider the results of both methods. This dual approach is assumed to be an effective tool as an accurate, high-throughput means of the screening of GMOs for potential genetic instabilities that may interfere with the detection and identification of specific GM events.

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This article does not contain any studies with human or animal subjects.

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Authors and Affiliations

  1. Austrian Agency for Health and Food Safety, Department for Molecular Biology and Microbiology, Institute for Food Safety Vienna, Spargelfeldstrasse 191, 1220, Vienna, Austria

    Zita Erika Madi, Christian Brandes & Rupert Hochegger

  2. GenØk—Centre for Biosafety, Forskningsparken, PB 6418, 9294, Tromsoe, Norway

    David Quist

  3. Austrian Agency for Health and Food Safety, Department for Medical Microbiology and Hygiene, Core Unit Molecular Biology, Spargelfeldstrasse 191, 1220, Vienna, Austria

    Werner Ruppitsch

  4. Baxter Innovations GmbH, Industriestraße 67, 1221, Vienna, Austria

    Georg Neumann

Authors
  1. Zita Erika Madi
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  2. Christian Brandes
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  3. Georg Neumann
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  4. David Quist
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  5. Werner Ruppitsch
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Correspondence to Christian Brandes.

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Madi, Z.E., Brandes, C., Neumann, G. et al. Evaluation of Adh1 alleles and transgenic soybean seeds using Scorpion PCR and HRM analysis. Eur Food Res Technol 237, 125–135 (2013). https://doi.org/10.1007/s00217-013-1969-y

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  • DOI: https://doi.org/10.1007/s00217-013-1969-y

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