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Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms

Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers’ perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

A broad multiplex real‐time PCR method for the detection and identification of GMOs

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Fig. 1

Abbreviations

CRM:

Certified reference material

GMO:

Genetically modified organisms

IPC:

Internal positive control

LOD:

Limit of detection

MGB:

Minor groove binder

NTC:

No template control

PCR:

Polymerase chain reaction

RTi-PCR:

Real-time PCR

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Acknowledgments

We would like to thank Frederic Aymes, Celeste Chia, Hui Zhen Ho and Yuying Zhong from Nestlé Quality Assurance Center, Singapore, for their valuable contribution, as well as Matthias Kiehne from Life Technologies for his support on this project.

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Correspondence to Geoffrey Cottenet.

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Cottenet, G., Blancpain, C., Sonnard, V. et al. Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms. Anal Bioanal Chem 405, 6831–6844 (2013). https://doi.org/10.1007/s00216-013-7125-5

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  • DOI: https://doi.org/10.1007/s00216-013-7125-5

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