A novel method for detection of HBVcccDNA in hepatocytes using rolling circle amplification combined with in situ PCR

Background: Intrahepatic hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is the original template for HBV replication. The persistence of cccDNA is responsible for the recurrence of HBV infection. The detection of cccDNA can help the development of new antiviral drugs against HBV replication links, and reduce the resistance and recurrence as well as to discover extrahepatic HBV infection. In situ polymerase chain reaction (IS-PCR) can be used to determine the distribution and localization of cccDNA in liver tissues, but it is hampered by its low sensitivity and specificity. We developed a novel method to detect HBV cccDNA using rolling circle amplification (RCA) combined with IS-PCR. Methods: Biopsy liver tissues were obtained from 26 patients with HBV infection, including 10 chronic hepatitis B (CHB), 6 liver cirrhosis (LC) and 10 hepatocellular carcinoma (HCC) patients. Four pairs of primers were designed to mediating RCA for the first round amplification of HBV cccDNA specifically. The liver tissue sections from patients were treated by plasmid-safe ATP-dependent DNase (PSAD) prior to RCA. After RCA, HBV cccDNA was further amplified by a pair of selective primers labeled digoxigenin that target the gap region between the two direct repeat regions (DR1 and DR2) of the virus via IS-PCR. Results: HBVcccDNA was expressed and located in hepatocyte nucleus in 19 patients (73.07%). Compared with the IS-PCR, the introduction of RCA increase the limit of detection. RCA combined with IS-PCR yielded strong positive signals in HCC liver tissue in spite of low copy number cccDNA (2 copies of target sequence per cell), meanwhile, no positive signal was detected via negative control. Conclusions: RCA combined with IS-PCR is an effective and practicable method which could detect the presence of low copy number of cccDNA sensitively and specifically, and reflect the relationship between cccDNA expression level and liver tissue pathological characteristics.