A CRISPR-based and post-amplification coupled SARS-CoV-2 detection with a portable evanescent wave biosensor

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biosensing

Background:

• Reliable diagnosis of SARS-CoV-2 is critical for public health during the ongoing COVID-19 pandemic.
• Existing diagnostic methods may face limitations in speed, ease of implementation, or sensitivity.

Purpose of the Study:

• To develop a rapid, accurate, and easy-to-implement CRISPR-based biosensing platform for SARS-CoV-2 detection.
• To achieve high sensitivity and specificity for point-of-care diagnosis of COVID-19.

Main Methods:

• Utilized CRISPR-Cas13a collateral activity combined with enzyme-free hybridization chain reaction (HCR) amplification.
• Designed a cleavage hairpin reporter to release an initiator sequence upon target recognition.
• Detected HCR amplicons via evanescent wave fluorescence on a desthiobiotin-modified optical fiber.
• Programmed three Cas13a crRNAs targeting S, N, and Orf1ab genes of SARS-CoV-2.

Main Results:

• Achieved rapid detection of SARS-CoV-2 in under one hour.
• Demonstrated attomolar sensitivity for target RNA detection.
• Validated the method using SARS-CoV-2 RNA in negative oropharyngeal swabs.
• Showcased broad detection capabilities for related coronaviruses (MERS-CoV, SARS-CoV).

Conclusions:

• The developed CRISPR-Cas13a/HCR biosensing platform offers a promising solution for rapid and sensitive COVID-19 diagnosis.
• The system's ease of implementation and high discrimination capability support its potential for point-of-care applications.
• This technology can aid in timely public health interventions and disease management.