Visual fluorescence detection of Listeria monocytogenes with CRISPR-Cas12a aptasensor

Area of Science:

• Food Microbiology
• Biosensing Technology
• Molecular Diagnostics

Background:

• Listeria monocytogenes is a significant foodborne pathogen causing listeriosis, a serious illness with potential fatal outcomes.
• Current detection methods for L. monocytogenes can be time-consuming and lack sensitivity for rapid on-site applications.
• There is a need for sensitive, specific, and rapid diagnostic tools for L. monocytogenes in food products.

Purpose of the Study:

• To develop and validate a novel aptasensor for the rapid and sensitive detection of Listeria monocytogenes.
• To utilize the Cas12a enzyme system in conjunction with aptamers for enhanced detection capabilities.
• To evaluate the performance of the aptasensor in terms of sensitivity, specificity, and applicability to real food samples.

Main Methods:

• Development of an aptasensor using carboxylated magnetic beads and the Cas12a enzyme.
• Exploitation of aptamer conformational changes upon binding to L. monocytogenes to trigger Cas12a trans-cleavage activity.
• Quantification of L. monocytogenes by measuring fluorescence intensity changes using a blue light gel cutter.

Main Results:

• The Cas12a aptasensor demonstrated high sensitivity for L. monocytogenes detection, with a limit of detection (LOD) as low as 57.15 CFU/mL.
• A wide linear detection range (4×10^2 to 4×10^7 CFU/mL, R²=0.9858) was achieved, indicating good quantitative performance.
• The aptasensor showed excellent selectivity and recovery rates in spiked beef samples, confirming its practical applicability.

Conclusions:

• The developed Cas12a aptasensor provides a novel, rapid, and visually detectable method for L. monocytogenes.
• This biosensor offers advantages in simplicity, speed, and stability compared to existing detection techniques.
• The aptasensor is suitable for on-site detection of L. monocytogenes in food matrices like beef, enhancing food safety surveillance.