A warm-start digital CRISPR/Cas-based method for the quantitative detection of nucleic acids

Area of Science:

• Molecular Diagnostics
• CRISPR-Cas Systems
• Nucleic Acid Detection

Background:

• CRISPR/Cas systems are emerging as rapid and sensitive tools for molecular diagnostics.
• Current CRISPR-based diagnostics often lack quantitative capabilities.
• Accurate nucleic acid quantification is crucial for various diagnostic applications.

Purpose of the Study:

• To develop a rapid, sensitive, and quantitative nucleic acid detection method using CRISPR/Cas.
• To evaluate the performance of the developed WS-RADICA method for various nucleic acid targets.
• To compare WS-RADICA with existing diagnostic techniques.

Main Methods:

• Development of Warm-Start RApid DIgital Crispr Approach (WS-RADICA).
• Utilized digital chips for DNA and RNA quantification.
• Assessed detection limits, dynamic ranges, and inhibitor tolerance.
• Validated WS-RADICA with SARS-CoV-2, human adenovirus, and herpes simplex virus.

Main Results:

• WS-RADICA achieved sensitive detection of SARS-CoV-2 RNA (1 copy/μl) in 40-60 minutes.
• Demonstrated broad linear dynamic ranges for DNA (0.8-12777 copies/μL) and RNA (1.2-18391 copies/μL) with high R² values.
• Showed lower detection limits and higher inhibitor tolerance compared to RT-LAMP-Cas12b.
• Exhibited comparable performance to RT-qPCR and RT-dPCR.

Conclusions:

• WS-RADICA provides a rapid, sensitive, and quantitative CRISPR-based diagnostic approach.
• The method is adaptable to digital devices and robust against inhibitors.
• WS-RADICA holds significant potential for diverse nucleic acid quantification applications, including pathogen detection.