An Accurate, Rapid and Cost-Effective Method for T-nos Detection Based on CRISPR/Cas12a

Area of Science:

• Molecular Biology
• Biotechnology
• Agricultural Science

Background:

• CRISPR/Cas12a is valuable for nucleic acid detection due to specific recognition and DNA cleavage.
• Existing methods for detecting genetically modified (GM) organisms can be costly and require specialized equipment.

Purpose of the Study:

• To develop a sensitive, rapid, and cost-effective fluorescence-based detection method for GM crops.
• To optimize a combined Polymerase Chain Reaction (PCR) and CRISPR/Cas12a system for detecting the nopaline synthase terminator (T-nos) gene.

Main Methods:

• Developed a fluorescence visualization detection assay integrating PCR amplification and CRISPR/Cas12a.
• Optimized PCR and CRISPR/Cas12a reaction systems for enhanced sensitivity and stability.
• Validated the method using 11 soybean samples, assessing detection limits and specificity.

Main Results:

• The PCR-CRISPR/Cas12a method achieved specific detection of target genes down to 60 copies within 50 minutes.
• Accurate detection was confirmed across all 11 tested soybean samples.
• The assay demonstrated high sensitivity and specificity for GM component detection.

Conclusions:

• A novel, accurate, rapid, and cost-effective method for GM detection was successfully developed.
• The method's portability and lack of need for large-scale instruments make it suitable for field-based mass detection.
• This technique offers a practical solution for monitoring transgenic materials in plants.