One-Step RAA and CRISPR-Cas13a Method for Detecting Influenza B Virus

Area of Science:

• Molecular Biology
• Virology
• Diagnostic Technologies

Background:

• Influenza B Virus (IBV) poses a significant public health challenge.
• Early and accurate diagnosis is crucial for limiting IBV transmission.
• Existing diagnostic methods may lack the speed or field applicability required for rapid response.

Purpose of the Study:

• To develop a sensitive and specific diagnostic method for early Influenza B detection.
• To create a rapid, field-deployable test for IBV nucleic acid.
• To evaluate the performance of the novel diagnostic method against established techniques.

Main Methods:

• Development of a CRISPR-based Rapid and Efficient Test (CRISPRET) utilizing recombinase-aided amplification (RAA) and CRISPR-Cas13a.
• Design of specific primers and CRISPR RNAs targeting conserved regions of the IBV NS gene.
• Optimization of the reaction system and validation using IBV nucleic acid reference materials and clinical samples.
• Comparative analysis with quantitative polymerase chain reaction (qPCR) for sensitivity, specificity, and predictive values.

Main Results:

• CRISPRET demonstrated a detection limit of 500 copies·μL^-1.
• The method achieved high diagnostic accuracy: 100% sensitivity, 97.69% specificity, 97.69% PPV, and 100% NPV.
• IBV nucleic acid detection occurred within approximately 5 minutes, with a total reaction time of 30 minutes.
• CRISPRET showed a high concordance rate of 98.83% with qPCR.

Conclusions:

• CRISPRET is a simple, rapid, and field-applicable one-step method for IBV detection.
• The assay exhibits high sensitivity and specificity, making it suitable for early diagnosis.
• CRISPRET has significant potential for integration into existing diagnostic systems and for field-testing applications to control IBV spread.