A CRISPR/Cas12a-hyperbranched rolling circle amplification sensor for ultrasensitive visual bacteria detection.

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Pathogenic bacteria spread rapidly through food, air, and water, posing significant public health risks.
• Conventional detection methods are often slow, require bulky equipment, and lack sensitivity for on-site screening.
• There is a critical need for rapid, sensitive, and visual detection platforms for pathogen surveillance.

Purpose of the Study:

• To develop a sensitive and visual pathogen detection platform for on-site quantification.
• To integrate CRISPR/Cas12a, hyperbranched rolling circle amplification (HRCA), and copper fluorescence nanoparticles (CuNPs) for enhanced signal amplification.
• To enable smartphone-based quantitative readout for point-of-need applications.

Main Methods:

• Constructed a Cas12a-HRCA detection platform integrating CRISPR/Cas12a, HRCA, CuNPs, and smartphone readout.
• Utilized pathogen target-activated CRISPR/Cas12a to regulate HRCA initiation, generating AT-TA-rich sequences.
• Employed CuNPs self-assembly on HRCA products for fluorescence signal amplification, coupled with RGB analysis via smartphone.

Main Results:

• Achieved a detection limit of 1 CFU/mL for *S. aureus* with high specificity.
• Demonstrated reliable recovery rates of 91%-106% in spiked milk samples without pre-enrichment or purification.
• The Cas12a-HRCA platform provided direct, on-site quantitative readout with integrated signal amplification.

Conclusions:

• The Cas12a-HRCA platform offers a sensitive, specific, and visual method for pathogen quantification.
• This technology shows great potential for point-of-need food safety monitoring and the detection of various pathogens in diverse settings.
• The integration of CRISPR/Cas12a and HRCA with smartphone readout provides a powerful tool for rapid diagnostics.