Digital CRISPR/Cas13a-RPA based assay for rapid and sensitive detection of JC virus in renal transplant patients

Abstract

JC viruses (JCVs), members of the Polyomaviruses family, are a type of circular double-stranded DNA (dsDNA) viruses and can lead to opportunistic infections. Infection of JCVs in kidney transplant patients can lead to polyomavirus-associated nephropathy. Thus, early detection and quantification of JC virus in urine or blood will effectively improve the survival rate of kidney transplant patients and reduce the mortality of patients. At present, PCR has been used as a routine method for JC virus detection in clinical practice. However, PCR cannot achieve absolute quantitative detection of viral nucleic acid. rendering it unsuitable for certain clinical applications with specific requirements. Herein, we proposed a JC virus nucleic acid testing platform, named MdCaR, which couples a microfluidic droplet device with CRISPR-Cas13a and recombinase polymerase amplification. MdCaR can quantitatively detect JC virus through digital droplet. The detection limit achieves 1 copy/mL, and the assay takes only 40 min. Twelve renal transplant patients and 20 control subjects’ specimens were analyzed. MdCaR conveyed a good performance in testing these samples, especially for detecting the low viral load samples identified as negative by qPCR. Taken altogether, MdCaR could be regarded as a promising, rapid JCV detection strategy and widely applied for diagnosis for JCV infection.