CRISPR/Cas12a-Based Versatile Method for Checking Quantitative Polymerase Chain Reaction Samples with Cycles of...

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostic Assays

Background:

• Quantitative polymerase chain reaction (qPCR) is a standard diagnostic tool, but generates ambiguous "gray zone" results requiring retesting.
• Repetitive qPCR testing for gray zone samples increases workload without guaranteeing resolution.
• CRISPR/Cas12a systems offer high specificity and signal amplification for nucleic acid detection.

Purpose of the Study:

• To develop and validate a CRISPR-based method for resolving gray zone samples from qPCR assays.
• To enhance the convenience and safety of CRISPR-based detection with a specialized cassette.
• To demonstrate the method's efficacy using African swine fever virus (ASFV) as a model.

Main Methods:

• Utilized CRISPR/Cas12a system for specific nucleic acid recognition and collateral ssDNA cleavage.
• Designed a gray zone checking cassette for convenient and contained CRISPR reaction assays.
• Applied the method to African swine fever virus (ASFV) detection in real swine blood samples.

Main Results:

• The CRISPR-based method successfully resolved all 6 ASFV qPCR gray zone samples.
• The entire checking process, including CRISPR reaction, was completed within 10-15 minutes.
• The method demonstrated high specificity and amplification, confirming its utility in practical diagnostics.

Conclusions:

• The CRISPR-based checking method provides an effective solution for eliminating gray zone issues in qPCR.
• The developed method is operable, applicable in practical settings, and complements existing PCR-based diagnostics.
• This approach offers a rapid and reliable supplementary tool for disease diagnosis, including potential applications for COVID-19.