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Development and application of a quantitative loop-mediated isothermal amplification method for detecting genetically

Sicong Huang1, Yuancong Xu, Xinghua Yan

  • 1High School attached to Tsinghua University, Beijing, 100084, China.

Journal of the Science of Food and Agriculture
|April 29, 2014
PubMed
Summary

A new SYBR Green I-based quantitative loop-mediated isothermal amplification (LAMP) assay enables rapid detection of genetically modified maize MON863. This sensitive and accurate method provides reliable results within one hour.

Keywords:
Cry3Bb1genetically modified maizeloop-mediated isothermal amplificationquantitative

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Food Safety

Background:

  • Genetically modified (GM) crops require reliable detection methods.
  • Maize MON863 contains specific gene insertions (Cry3Bb1 and tahsp17).
  • Existing detection methods may lack speed or sensitivity.

Purpose of the Study:

  • To develop a rapid and sensitive quantitative assay for detecting GM maize MON863.
  • To optimize conditions for a SYBR Green I-based loop-mediated isothermal amplification (LAMP) assay.

Main Methods:

  • Design of primers targeting the integration region of Cry3Bb1 and tahsp17 genes in maize MON863.
  • Optimization of reaction conditions including dNTPs, betaine, primers, Mg(2+), Bst polymerase, temperature, and time.
  • Utilizing SYBR Green I for real-time fluorescence detection during isothermal amplification.

Main Results:

  • Optimized reaction conditions identified critical roles for Mg(2+) and betaine concentrations.
  • Achieved a low detection limit of 4 haploid genomic DNA copies for both qualitative and quantitative LAMP.
  • Demonstrated completion of LAMP reactions within 1 hour at 65 °C.

Conclusions:

  • The developed SYBR Green I-based quantitative LAMP assay is reliable, sensitive, and accurate.
  • This method offers a rapid and efficient tool for detecting genetically modified maize MON863.
  • The assay holds potential for routine application in food safety and agricultural monitoring.