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Fast and Accurate Multiplex Identification and Quantification of Seven Genetically Modified Soybean Lines Using

Alexandra Bogožalec Košir1, Sabine Muller2, Jana Žel1

  • 1Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 121, 1000 Ljubljana, Slovenia.

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Summary

Digital PCR (dPCR) enables multiplexing to quantify multiple genetically modified organisms (GMOs) simultaneously. This study demonstrates a six-color dPCR platform for precise GMO soybean detection in complex samples, improving testing efficiency.

Keywords:
6-color systemdigital PCRgenetically modified organismsmultiplexingquantification

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Area of Science:

  • Agricultural Biotechnology
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Genetically modified organisms (GMOs) require accurate quantification for regulatory and safety purposes.
  • Traditional quantitative real-time PCR (qPCR) struggles with the multiplex detection of numerous GMOs in single samples.
  • Digital PCR (dPCR) offers enhanced multiplexing capabilities for precise GMO quantification.

Purpose of the Study:

  • To evaluate the Naica six-color Crystal dPCR platform for simultaneous quantification of multiple GM soybean lines.
  • To develop and validate flexible multiplex assays for GMO detection.
  • To optimize data analysis methods for improved limit of detection (LOD) and limit of quantification (LOQ).

Main Methods:

  • Development of six-color and four-color multiplex dPCR assays for GM soybean quantification.
  • Assessing assay specificity, sensitivity (LOD), and precision (bias).
  • Implementing limit-of-blank (LOB) correction and reaction pooling for enhanced sensitivity.

Main Results:

  • High specificity, sensitivity (LOD < 25 copies/reaction), and precision (bias < 15%) were achieved for the dPCR assays.
  • LOB correction refined LOQ and LOD determination.
  • Reaction pooling demonstrated a two- to eight-fold increase in sensitivity, lowering the LOD.
  • Assays were successfully validated on real-life samples from routine GMO testing.

Conclusions:

  • The six-color Crystal dPCR platform effectively quantifies multiple GM soybean lines in a single assay.
  • Optimized data analysis, including LOB correction and pooling, enhances assay performance.
  • This dPCR approach offers a powerful tool to revolutionize GMO testing, enabling comprehensive analysis of complex samples.