A CRISPR-Cas13a-based two-step assay combined with lateral flow strips for rapid detection of Epstein-Barr virus

Epstein-Barr virus (EBV) is a widespread oncogenic virus associated with multiple human malignancies. Accurate detection of EBV DNA is essential for disease screening and monitoring; however, commonly used methods such as quantitative PCR require specialized instruments and laboratory infrastructure, limiting their applicability in resource-limited settings.

An isothermal nucleic acid detection method for EBV DNA was developed by combining recombinase-aided amplification with CRISPR/Cas13a detection. Target DNA was amplified under constant temperature conditions. Subsequently, Cas13a-mediated collateral RNase activity triggered the cleavage of labeled reporter molecules to produce detectable signals. Fluorescence measurement and lateral flow strip analysis were used for result interpretation.

The fluorescence assay detected EBV DNA across a dynamic range up to 10⁷ copies per reaction. The limit of detection (LoD) was established at 10¹ copies per reaction. No cross-reactivity was observed. The lateral flow strip assay showed visible positive results at concentrations of 10⁴ copies per reaction and higher. In the preliminary feasibility evaluation using clinical blood samples, the fluorescence and lateral flow assays achieved positive detection rates of 100 and 84.6% (11/13), respectively, with both methods demonstrating no cross-reactivity against the tested control viruses.

This study developed a two-step RAA-CRISPR/Cas13a assay for EBV DNA detection. The assay combines analytical sensitivity with an instrument-free visual readout. These findings demonstrate the technical feasibility of the RAA-CRISPR platform for EBV screening; however, further optimization is required to enhance the sensitivity of the visual readout. Large-scale clinical studies are essential to fully determine its diagnostic performance and practical utility in decentralized and resource-limited settings.