A calibrator plasmid for quantitative analysis of insect resistant maize (Yieldgard MON 810)

Area of Science:

• Biotechnology
• Molecular Biology
• Food Science

Background:

• Real-time PCR (RT-PCR) is crucial for quantifying genetically modified organisms (GMOs) and enforcing labeling regulations.
• The accuracy of RT-PCR relies heavily on the quality of external calibrators used.
• Conventional calibrants like seed powder can present challenges in precision and stability.

Purpose of the Study:

• To construct and validate a novel dual-target plasmid, pRSETMON-02, for quantifying MON 810 maize.
• To assess the commutability and performance of plasmid DNA (pDNA) versus genomic DNA (gDNA) calibrants in RT-PCR.
• To evaluate the stability of the developed pDNA calibrant.

Main Methods:

• Construction and validation of a dual-target plasmid (pRSETMON-02) containing MON 810 maize event-specific and zein gene sequences.
• Assessment of pDNA and gDNA calibrants using TaqMan RT-PCR targeting the P-35S promoter and zein gene.
• Evaluation of PCR efficiency, linearity, relative standard deviation (RSD), and stability of calibrants.

Main Results:

• The pRSETMON-02 plasmid demonstrated higher PCR efficiencies, better linearity, and lower RSD compared to gDNA calibrants.
• pDNA calibrants showed superior performance in accurately quantifying unknown samples.
• The pRSETMON-02 plasmid exhibited good short-term stability, remaining stable for 45 days at -20°C and 4°C.

Conclusions:

• The developed dual-target plasmid pRSETMON-02 is a suitable and effective calibrant for quantifying MON 810 maize.
• pRSETMON-02 offers a more reliable and reproducible alternative to traditional seed powder calibrants for GMO quantification.
• This advancement supports accurate GMO detection and regulatory compliance.