Sensitive and specific CRISPR-Cas12a assisted nanopore with RPA for Monkeypox detection

Area of Science:

• Molecular Diagnostics
• Infectious Disease Detection
• CRISPR Technology

Background:

• Monkeypox virus (MPXV) outbreaks necessitate rapid, accurate diagnostic tools for effective public health response.
• Existing diagnostic methods may face limitations in sensitivity, speed, or point-of-care applicability.
• Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology offers a promising avenue for next-generation molecular diagnostics.

Purpose of the Study:

• To develop a highly sensitive and specific diagnostic method for Monkeypox virus (MPXV) detection.
• To create a point-of-care applicable assay overcoming limitations of existing PCR-based CRISPR methods.
• To evaluate the performance of a novel CRISPR-Cas12a assisted Nanopore (SCAN) system integrated with isothermal Recombinase Polymerase Amplification (RPA).

Main Methods:

• Development of a CRISPR-Cas12a assisted Nanopore (SCAN) system combined with isothermal Recombinase Polymerase Amplification (RPA).
• Utilized size-counting of single molecules to analyze reporter cleavage kinetics.
• Assessed the limit of detection (LoD) and specificity of the RPA-SCAN assay for MPXV detection.

Main Results:

• The MPXV-specific RPA assay achieved a limit of detection (LoD) of 19 copies in a 50 μL reaction.
• The integrated RPA-SCAN system demonstrated an overall LoD of 16 copies/μL (26.56 aM) for MPXV.
• The RPA-SCAN method showed 100% accuracy in distinguishing MPXV from cowpox virus, confirming high specificity.

Conclusions:

• The isothermal RPA-SCAN device provides a highly sensitive and specific method for Monkeypox detection.
• This system overcomes limitations of PCR-SCAN, making it suitable for point-of-care applications.
• The miniaturization potential of RPA-SCAN positions it as a versatile platform for diagnosing various infectious diseases.