Photoactivated Digital Recombinase Polymerase Amplification/CRISPR-Cas12a Assay for Point-of-Care of BK Polyomavirus Quantification.

Abstract

BK polyomavirus (BKV) serves as a critical biomarker for optimizing immunosuppressive therapy and preventing graft failure in kidney transplant recipients. Quantitative PCR (qPCR), the current gold standard for BKV load quantification, relies on batch-specific standard curves. For kidney transplant recipients, this not only elevates the risk of cross-contamination but also entails considerable economic burdens. Therefore, developing BKV quantification technologies independent of batch-specific standard curves is of great clinical significance for this patient population. The combination of CRISPR-Cas12a with recombinase polymerase amplification (RPA), termed DETECTR, offers notable advantages for digital nucleic acid analysis. However, due to the high viscosity of RPA reagents, the generation of high-throughput, uniform RPA microdroplets remains a significant technical challenge. In the present study, we developed a centrifugal RPA microdroplet generation method based on commercial capillaries, facilitating the production of high-throughput, uniform RPA microdroplets (23.1 μm in diameter) via simple centrifugation. Furthermore, by integrating a light-controlled RPA-CRISPR-Cas12a system, we established photoactivated digital DETECTR (pd-DETECTR) for precise, point-of-care, and cost-effective BKV quantification. When combined with a smartphone-based reader, the pd-DETECTR assay can be completed within 42 min. Clinical validation demonstrated a strong correlation (<i>R</i>² = 0.9801) with qPCR results, exhibiting high sensitivity (100.0%), specificity (98.0%), and accuracy (99.0%). The pd-DETECTR provides a rapid, convenient, and cost-effective tool for BKV load analysis, which can significantly reduce the economic burden and risk of opportunistic infections in kidney transplant recipients, thus holding significant clinical value.