To establish a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (CRISPR/Cas13a)-based detection method for hepatitis B virus covalently closed circular DNA (HBV cccDNA).

After extracting the total DNA from the livers of 4 patients with hepatitis B collected in Beijing You’an Hospital, Capital Medical University from June 2017 to October 2020, total DNA was digested with Hind Ⅲ endonuclease and plasmid-safe ATP-dependent DNase (PSAD), respectively; According to the structural differences between relaxed circular DNA (rcDNA) and cccDNA, primers for specific amplification of HBV cccDNA were designed, and the products after digestion were subjected to rolling circle amplification (RCA) and PCR amplification; And crRNA was screened to establish a new method for HBV cccDNA detection based on CRISPR/Cas13a technology.

Alpha-1 antitrypsin (A1AT) and hepatitis B virus surface antigen (HBsAg) primers were used to amplify the double digested product to verify the existence of hepatitis B virus genome in the product; Using HBV cccDNA and HBV rcDNA primers to amplify the product after PSDA digestion, it was verified that only HBV cccDNA exists in the product. The positive sample after RCA was used as a template for gradient dilution, and then PCR amplification was performed and CRISPR/Cas13a detection was used after transcription. The lower limit of detection was calculated to be 10 copies/μl.

A novel detection method of RCA-PCR-CRISPR-Cas13a was established, which can detect HBV cccDNA with high sensitivity and high specificity, and provide an effective monitoring method for the evaluation of antiviral therapy of hepatitis B patients, the determination of treatment endpoints, and the adjustment of treatment plans.