CRISPR/Cas13a-assisted rapid and portable HBV DNA detection for low-level viremia patients

Abstract

Background & Aims: The WHO declared to eliminate hepatitis B virus (HBV) by 2030. However, an increasing number of patients are presenting with low-level viremia (LLV) with the widespread use of antiviral medications. The diagnostic efficiency and coverage area of HBV infection are low. Hence, this study intended to drive the HBV infection detection to effectively adaptable for any small to medium-sized laboratory or field survey. Methods: We established, optimized, and evaluated a colloidal gold test strip for detection of HBV DNA based on CRISPR/Cas13a combined with recombinase-aided amplification (RAA) technology. Furthermore, 180 HBV-infected patients (including patients with different viral loads, LLV patients and dynamic plasma samples of patients on antiviral therapy) were enrolled for clinical validation. Results: The strip detection of HBV DNA was established based on RAA-CRISPR-Cas13a technology with a sensitivity of 10 1 copies/μL and a specificity of 100%. HBV DNA gradient concentration plasmids and clinical samples were effectively identified by this approach. The positive coincidence rate for LLV patients was 87%, while the negative coincidence rate was 100%. The positive coincidence rate reached 100% in LLV patients (viral loading >100 IU/mL). The sensitivity, specificity, positive predictive agreement (PPA) and negative predictive agreement (NPA) values of dynamic plasma detection in patients on antiviral therapy were 100%, 92.15%, 93.75%, and 100%, respectively. Conclusions: We develop rapid and portable RAA-CRISPR/Cas13a-based strip of HBV DNA detection for LLV patients. This study provides a visual and faster alternative to current PCR-based diagnosis for HBV infection.

A spacer design strategy for CRISPR‐Cas12f1 with single‐nucleotide polymorphism mutation resolution capability and its application in the mutations diagnosis of pathogens

“…Rapid, highly accurate, and sensitive nucleic acid detection platforms were developed using CRISPR‐Cas system 11,26–29 . However, the resolution of CRISPR‐Cas tools in SNP identification remains to be addressed 30 .…”

Cas-based bacterial detection: recent advances and perspectives

“…If a bacterium is infected by a bacteriophage, it can activate programmed cell death or enter a dormant state to limit the spread of infection within the entire population. The excellent detection capability of CRISPR/ Cas13a has been applied to detect H7N9, 71 CPV-2, 72 HBV, 73 and many other viruses.…”

Application of CRISPR/Cas13a-based biosensors in serum marker detection

“…Subsequently, they developed a colloidal gold test strip combined with RAA and Cas13a, which was used to detect HBV DNA in the plasma with hypoxemia, with a sensitivity of 10 copies per μL. 53 A highly sensitive biosensor called the PCR-based CRISPR/Cas13a detection system (PCR-CRISPR) has been established to detect HBV DNA and drug resistance, which can be detected within 15 minutes after the end of PCR amplification. 54…”