Validation of a Qualitative Detection Method for Influenza A Virus RNA Based on the RT-RPA-CRISPR/Cas13a System

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Seasonal influenza A virus (IAV) outbreaks necessitate rapid, cost-effective detection methods.
• Current diagnostics like RT-qPCR, antigen tests, and existing CRISPR platforms have limitations in sensitivity, complexity, or cost.
• There is a critical need for accessible and reliable IAV diagnostic tools, particularly for resource-limited environments.

Purpose of the Study:

• To develop a simplified, equipment-free platform for qualitative detection of influenza A virus.
• To adapt Reverse Transcription Recombinase Polymerase Amplification (RT-RPA) and CRISPR-Cas13a for sensitive and specific IAV detection.
• To validate the performance of the developed platform using clinical samples.

Main Methods:

• Integrated isothermal Reverse Transcription Recombinase Polymerase Amplification (RT-RPA) with CRISPR-Cas13a for nucleic acid amplification and detection.
• Utilized a lateral flow strip for visual readout, simplifying result interpretation.
• Employed a rapid nucleic acid release reagent for streamlined sample preparation.

Main Results:

• The platform demonstrated high sensitivity, detecting as low as 10¹ copies/mL of viral genome.
• Achieved 100% concordance with RT-qPCR when tested on 78 clinical samples.
• Showed no cross-reactivity with other common respiratory viruses, confirming specificity.

Conclusions:

• The simplified RT-RPA-CRISPR/Cas13a workflow provides a sensitive, specific, and equipment-free method for influenza A virus detection.
• This innovative diagnostic approach is suitable for rapid testing in low-resource settings, addressing a significant public health need.
• The platform's performance suggests its potential for widespread implementation in point-of-care diagnostics.