Adsorption-Free Self-Priming Direct Digital Dual-crRNA CRISPR/Cas12a-Assisted Chip for Ultrasensitive Detection of...

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Microfluidics

Background:

• Rapid and sensitive pathogen detection is crucial for disease diagnosis and treatment.
• RPA-CRISPR/Cas12 systems show promise for pathogen detection, but face challenges in microfluidic applications.
• Existing self-priming digital PCR chips struggle with protein adsorption and multi-step processes.

Purpose of the Study:

• To develop an adsorption-free self-priming digital chip.
• To establish a direct digital dual-crRNAs (3D) assay for ultrasensitive pathogen detection.
• To enable rapid and accurate quantification of Salmonella in point-of-care settings.

Main Methods:

• Developed an adsorption-free self-priming digital microfluidic chip.
• Established a direct digital dual-crRNAs (3D) assay integrating RPA, CRISPR/Cas12a, and digital PCR.
• Targeted the invA gene of Salmonella for detection.

Main Results:

• Achieved ultrasensitive detection of Salmonella with a limit of detection of approximately 0.2 cells/mL.
• Demonstrated a linear detection range from 2.58 × 10^1 to 2.58 × 10^4 cells/mL.
• Successfully detected Salmonella directly in milk samples within 30 minutes without nucleic acid extraction.

Conclusions:

• The 3D assay on the adsorption-free chip provides accurate and reliable digital absolute quantification of Salmonella.
• This method offers a powerful nucleic acid detection platform for point-of-care testing.
• The developed assay facilitates the application of CRISPR/Cas-assisted detection and microfluidic chips for rapid pathogen identification.