End-point dual specific detection of nucleic acids using CRISPR/Cas12a based portable biosensor

Area of Science:

• Biotechnology
• Biosensor Development
• Molecular Diagnostics

Background:

• Genetically modified (GM) crops require accurate detection methods for regulatory compliance and consumer safety.
• Existing detection methods can be complex, time-consuming, and require specialized laboratory equipment.
• There is a need for rapid, portable, and sensitive biosensors for on-site GM ingredient analysis.

Purpose of the Study:

• To develop a CRISPR/Cas12a-based portable biosensor (Cas12a-PB) for simultaneous visual detection of specific genes in GM soybean.
• To integrate the Cas12a-PB with different amplification techniques for enhanced convenience and portability.
• To evaluate the sensitivity and specificity of the developed biosensor for GM ingredient detection.

Main Methods:

• Development of a polymethylmethacrylate (PMMA)-based portable biosensor with integrated detection chambers.
• Utilizing CRISPR/Cas12a technology for collateral cleavage and fluorescent signal generation.
• Combining the biosensor with Polymerase Chain Reaction (PCR) and Loop-Mediated Isothermal Amplification (LAMP) for nucleic acid amplification.
• Visual detection of target genes (CaMV35S promoter and Lectin gene) via green fluorescence under UV light.

Main Results:

• The Cas12a-PB successfully achieved simultaneous visual detection of CaMV35S promoter and Lectin gene in GM soybean.
• Detection limits as low as 0.1% transgenic ingredients were achieved.
• High specificity was confirmed using various GM and non-GM samples, including maize and soybean.
• Integration with rapid PCR and LAMP simplified the detection process and enhanced portability.
• A further optimized design eliminated the need for reaction tubes and mineral oil, simplifying operation.

Conclusions:

• The developed CRISPR/Cas12a portable biosensor (Cas12a-PB) offers a reliable and convenient solution for dual-target GM detection.
• The biosensor's portability and visual output make it suitable for on-site food safety assessments.
• This technology has potential applications in clinical diagnostics and food safety analysis for multiple target detection.