An Integrated One-Tube RPA-CRISPR/Cas13d Assay Coupled with Lateral Flow for Rapid PRRSV-1 Detection

Area of Science:

• Molecular Biology
• Biotechnology
• Infectious Disease Diagnostics

Background:

• Rapid and scalable diagnostic technologies are crucial for controlling infectious diseases.
• Current methods often require complex laboratory equipment and trained personnel, limiting their use in resource-limited settings.
• There is a need for user-friendly, rapid, and accurate diagnostic tools for point-of-care testing (POCT).

Purpose of the Study:

• To present the Single-Tube Extraction-free Platform (STEP), a streamlined CRISPR-based system for viral RNA detection.
• To demonstrate STEP's capabilities in terms of speed, sensitivity, accuracy, and user-friendliness for POCT.
• To evaluate STEP's performance in clinical settings compared to established diagnostic methods.

Main Methods:

• STEP integrates isothermal Recombinase Polymerase Amplification (RPA) with CRISPR-Cas13d-based nucleic acid detection.
• The platform utilizes lab-free extraction reagents and lyophilized formulations for enhanced stability and ease of use.
• Multimodal readouts include lateral flow strips, in-tube fluorescence, and fluorescence quantification.

Main Results:

• STEP provides a sample-to-answer workflow within 35 minutes.
• Clinical evaluation demonstrated 100% sensitivity and specificity compared to RT-qPCR.
• The platform operates effectively at ambient and body temperatures, minimizing equipment constraints.

Conclusions:

• STEP offers a robust, equipment-minimal platform for rapid, sensitive, and accurate viral RNA detection.
• Its user-friendly design, stability, and minimal instrumentation make it suitable for decentralized infectious disease diagnostics.
• STEP facilitates rapid public health responses and enhances capabilities for point-of-care testing.