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Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection

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Abstract

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.

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Acknowledgment

This study was financially supported by the European Commission through the Integrated Project Co-Extra, Contract No. 007158, under the 6th framework programme, priority 5, food quality and safety and Slovenian research agency programme Plant biotechnology and systems biology P4-0165. For help with graphical illustrations we kindly thank Damijan Gašparič, MArch.

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Authors and Affiliations

  1. Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia

    Meti Buh Gašparič, Jana Žel & Kristina Gruden

  2. Section for food bacteriology and GMO, National Veterinary Institute, P.O. Box 750, Sentrum, 0106, Oslo, Norway

    Torstein Tengs & Arne Holst-Jensen

  3. Consorci CSIC-IRTA and IBMB-CSIC, Jordi Girona 18-26, 08034, Barcelona, Spain

    Jose Luis La Paz, Maria Pla & Teresa Esteve

Authors
  1. Meti Buh Gašparič
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  2. Torstein Tengs
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  3. Jose Luis La Paz
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  4. Arne Holst-Jensen
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  5. Maria Pla
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  6. Teresa Esteve
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  7. Jana Žel
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  8. Kristina Gruden
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Correspondence to Meti Buh Gašparič.

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Buh Gašparič, M., Tengs, T., La Paz, J.L. et al. Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection. Anal Bioanal Chem 396, 2023–2029 (2010). https://doi.org/10.1007/s00216-009-3418-0

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  • DOI: https://doi.org/10.1007/s00216-009-3418-0

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