Rapid visual detection of phytase gene in genetically modified maize using loop-mediated isothermal amplification...

Area of Science:

• Biotechnology
• Molecular Biology
• Agricultural Science

Background:

• Genetically modified (GM) maize expressing high phytase activity was approved in China in 2009.
• Accurate detection of GM traits is crucial for regulatory compliance and food safety.

Purpose of the Study:

• To develop and validate a rapid, sensitive, and visual method for detecting the phytase gene in GM maize.
• To compare the performance of the developed method against conventional polymerase chain reaction (PCR).

Main Methods:

• Development of a highly specific loop-mediated isothermal amplification (LAMP) assay targeting the phytase gene.
• Optimization of reaction conditions for speed and visual detection.
• Sensitivity and quantitative analysis using varying concentrations of phytase genomic DNA.
• Comparison with conventional PCR coupled with gel electrophoresis.

Main Results:

• The LAMP assay detected the phytase gene in less than 20 minutes with visible amplification, eliminating the need for gel electrophoresis.
• The detection sensitivity was approximately 30 copies of phytase genomic DNA, significantly higher than conventional PCR.
• Quantitative analysis demonstrated a strong linear correlation between DNA copy number and time to result (Tt) values over a wide dynamic range (6x10^1 to 6x10^7 copies).
• The limit of quantification for the LAMP method was determined to be 60 copies.

Conclusions:

• The developed LAMP method is a visual, rapid, and highly sensitive tool for detecting the phytase gene in GM maize.
• Its simplicity and lack of requirement for specialized equipment make it ideal for field testing and high-throughput screening of GMO feeds.
• This method offers a significant advancement over traditional PCR techniques for GMO detection.