Event-specific qualitative and quantitative detection of five genetically modified rice events using a single...

Area of Science:

• Agricultural Biotechnology
• Molecular Biology
• Food Safety

Background:

• Accurate detection of genetically modified (GM) rice is crucial for regulatory compliance and food safety.
• Existing methods for identifying specific GM rice events can be challenging and require robust reference materials.
• The development of standardized controls is essential for reliable qualitative and quantitative analysis.

Purpose of the Study:

• To construct a novel standard reference plasmid, pUC-RICE5, for the detection of five specific GM rice events.
• To develop and validate a quantitative PCR (qPCR) assay using pUC-RICE5 for precise GM rice event detection.
• To establish a reliable method for routine identification of GM rice in food and agricultural products.

Main Methods:

• Construction of the pUC-RICE5 plasmid containing rice endogenous reference gene (sucrose phosphate synthase) and fragments of five GM rice events (Bt63, Kemingdao1, Kefeng6, Kefeng8, LLRice62).
• Modification of a qualitative PCR assay into a quantitative PCR (qPCR) method utilizing pUC-RICE5 as a positive control and calibrator.
• Validation of the qPCR assay for specificity, linearity (R² values 0.993–1.000), and repeatability (RSD 0.10%–0.67%).

Main Results:

• The pUC-RICE5 plasmid successfully served as a standard reference for event-specific detection.
• The developed qPCR assay demonstrated high accuracy and precision, with limits of detection around 1-10 copies of rice haploid genomes.
• The method achieved excellent linearity and repeatability, indicating its robustness for quantitative analysis.

Conclusions:

• The novel pUC-RICE5 plasmid is a valuable tool for the accurate qualitative and quantitative detection of five specific GM rice events.
• The validated qPCR assay provides a reliable and routine method for identifying GM rice, meeting regulatory standards.
• This advancement supports enhanced food safety monitoring and regulatory oversight of genetically modified rice