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 (R2 = 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.
Wu et al. (2026) studied this question.