Simultaneous and ultrasensitive detection of HBV DNA and RNA in CHB by utilizing Cas12a/Cas13a-based dual-target strategy

Simultaneous detection of low levels of serum HBV DNA plus RNA in HBV-infected patients receiving antiviral therapy is an effective strategy to evaluate the antiviral effect. In this research, we established an ultrasensitive dual PCR-CRISPR fluorescent assay (DPCFS) based on CRISPR-Cas12a/Cas13a system to simultaneous detection of low levels of HBV DNA and RNA. We optimized the DPCFS assay and evaluated the specificity and sensitivity employing the HBV plasmid. Clinical validation and comparison with commercial HBV DNA ultrasensitive qPCR assay or reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) were conducted using 46 serum samples of HBV-infected patients including 22 treatment-naïve and 24 nucleotide/nucleoside analogs (NAs)-treated patients. The limit of detection (LOD) for DPCFS platform was 5.8 copies/μL HBV DNA and 1.4 copies/μL HBV RNA, respectively, and the detection results could be directly visible to the naked eye under blue light without the need for any specialized instruments. Furthermore, clinical validation has 97.06 % sensitivity and 100 % specificity compared with commercial HBV DNA ultrasensitive qPCR assay. For serum HBV RNA detection, the sensitivity of DPCFS assay was 86.84 %, and the specificity was 87.5 % compared with RT-ddPCR. We developed a simultaneous and ultrasensitive detection of lower levels of HBV DNA and RNA in HBV-infected patients receiving antiviral therapy, which provide more accurate and simple strategy to evaluate the antiviral effect and treatment guidance.