CRISPR-based assays for the detection of BK virus and JC virus infections post-kidney transplantation.

BACKGROUND

Organ transplantation recipients encounter significant risks from acute or chronic infections that threaten graft survival. BK virus (BKV) and JC virus (JCV) are two prominent opportunistic infection viruses, and they may cause polyomavirus-associated nephropathy and graft kidney loss in patients who are in an immunosuppressed state after kidney transplantation. Hence, timely detection and sustained monitoring of the viral load are indispensable. However, the current diagnostic methods remain limited, and the development of new molecular detection technology is extremely urgent.

METHODS

The sequences and concentrations of clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA), the concentration of Cas13a, and the primers for recombinase polymerase amplification (RPA) were optimized for BKV and JCV detection. Next, a novel microfluidic dual-droplet chip was designed and fabricated, and it was integrated with CRISPR (ddCRISPR) to simultaneously qualitatively detect BKV and JCV. Subsequently, the ddCRISPR assay was verified using clinical samples. Then, a lateral flow strip combined with CRISPR (LFCRISPR) was developed for the detection of BKV and JCV in resource-limited settings.

RESULTS

A one-pot RPA-CRISPR reaction system was established and optimized for BKV and JCV detection. ddCRISPR can simultaneously and rapidly detect BKV and JCV with high sensitivity (10 copies/ml for BKV and 1 copy/ml for JCV), and provide absolute quantification, which is suitable for viral load detection and conducive to personalized and precise treatment for organ transplant recipients. LFCRISPR simplified the operational process through a simple visual readout, facilitating virus screening after organ transplantation.

CONCLUSIONS

These platforms incorporate molecular testing into the transplantation treatment model, thereby reducing costs, prolonging the survival time of the graft, improving the clinical outcomes of postoperative management in kidney transplantation, and enhancing the patients' quality of life.