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

Oct 9, 2025Journal of agricultural and food chemistry

Rapid detection of PRRSV-1 using a combined one-step genetic test with easy-to-read results

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CRISPR Gene Editing on OpenScience ↗PubMed ↗DOI ↗

Abstract

STEP achieves 100% sensitivity and specificity compared to RT-qPCR in detecting viral RNA within 35 minutes.

STEP is a diagnostic platform that integrates isothermal amplification with CRISPR technology for viral RNA detection.
The system offers multiple readout options, including lateral flow strips and fluorescence quantification.
Lab-free reagents and lyophilized formulations enhance usability and stability for point-of-care testing.
The platform can function at both ambient and body temperatures, minimizing equipment needs.
Clinical evaluation confirms STEP's effectiveness in providing rapid and accurate diagnostic results.

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Rapid and scalable diagnostic technologies are essential for controlling infectious diseases. We present STEP (Single-Tube Extraction-free Platform for CRISPR/Cas13d detection), a streamlined, equipment-minimal CRISPR-based platform enabling rapid, sensitive, and accurate viral RNA detection. STEP integrates isothermal RPA amplification with CRISPR-based nucleic acid cleavage, providing multimodal readouts including lateral flow strips, in-tube fluorescence, and fluorescence quantification. Lab-free extraction reagents and lyophilized formulations enhance user friendliness of STEP and stability for point-of-care testing (POCT), reduce cost, and eliminate cold-chain requirements. Optimization of baseline time and the ability to operate at both ambient and body temperatures minimize temporal and equipment constraints. Clinical evaluation showed 100% sensitivity and specificity versus RT-qPCR, delivering a sample-to-answer workflow within 35 min. STEP provides a robust platform for decentralized infectious disease diagnostics and rapid public health response, combining speed, user friendliness, and minimal instrumentation requirements.