A One-Pot CRISPR/Cas12a-Based Platform for Contamination-Free Nucleic Acid Amplification Detection

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostic Assays

Background:

• CRISPR-Cas12a offers rapid and specific detection of nucleic acids.
• Existing CRISPR-Cas12a methods for PCR/LAMP products often involve open-tube steps, increasing cross-contamination risk.
• Carryover contamination is a significant challenge in molecular diagnostics.

Purpose of the Study:

• To develop a novel one-pot reaction system for CRISPR-Cas12a-based detection of PCR/LAMP products.
• To eliminate carryover contamination in nucleic acid amplification assays.
• To facilitate endpoint detection with enhanced specificity and reduced risk of false positives.

Main Methods:

• Designed PCR/LAMP primers incorporating the protospacer-adjacent motif (PAM) site (TTT).
• Utilized CRISPR-Cas12a pre-incubation with specific crRNAs (crRNA1 and crRNA2) for targeted degradation of carryover DNA.
• Developed a one-pot system with a pre-stored Cas12a-detection complex for visual endpoint detection.

Main Results:

• Achieved efficient cleavage of cross-contaminating DNA while preserving the target gene due to PAM site incorporation.
• Demonstrated degradation of up to 10^6 copies of carryover contaminants within one hour.
• Enabled direct visual detection of amplified products using a fluorescent probe and a blue LED instrument.

Conclusions:

• The developed one-pot CRISPR-Cas12a system effectively prevents carryover contamination in PCR/LAMP assays.
• This method offers a robust solution for sensitive and specific nucleic acid detection in complex biological samples.
• The system shows significant potential for improving the reliability of molecular diagnostic platforms.