CRISPR-Cas13a/Cas12a Assisted Dual Portable and Visualized HDV and HBV Detection

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Hepatitis B virus (HBV) and hepatitis D virus (HDV) co-infection exacerbates liver disease progression.
• Current diagnostics lack a combined method for detecting both HBV and HDV nucleic acids simultaneously.

Purpose of the Study:

• To develop a sensitive and specific dual detection assay for HDV RNA and HBV DNA.
• To establish a CRISPR-Cas-based system for simultaneous detection of both viruses.

Main Methods:

• Developed a dual detection method using CRISPR-Cas12a with recombinase polymerase amplification (RAA) for HBV DNA and CRISPR-Cas13a with RT-RAA for HDV RNA.
• Optimized RAA primers and crRNAs for dual fluorescence (DF) and lateral flow strip (DL) detection.
• Validated the assay using synthetic samples and plasma from 70 co-infected patients.

Main Results:

• The CRISPR-Cas system demonstrated 100% specificity.
• Both DF and DL methods achieved a sensitivity of 10 copies/μL for synthetic samples.
• Positive concordance rates in clinical samples were 85.7% for DF and 82.9% for DL.

Conclusions:

• A CRISPR-Cas13a/Cas12a-based dual assay provides sensitive, specific, and accurate detection of HDV RNA and HBV DNA.
• This assay is a valuable tool for early detection, treatment, and monitoring of HBV/HDV co-infections.