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

Abstract

BackgroundIntrahepatic 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.MethodsBiopsy 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.ResultsHBVcccDNA 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.ConclusionsRCA 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0608-y) contains supplementary material, which is available to authorized users.

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

“…However, the detection of viral nucleic acids in almost all hepatocytes raised concerns regarding the diffusion of amplified DNA to neighboring cells, which had been a major issue with in situ PCR (22). Recently, a method using rolling circle amplification (RCA), combined with in situ PCR, was reported to detect HBV cccDNA (23). However, RCA reaction for the detection of DNA in formalinfixed tissues would presumably encounter serious problems, since cross-linked histones or other cccDNA-binding proteins would hinder effective amplification.…”

A new model mimicking persistent HBV e antigen-negative infection using covalently closed circular DNA in immunocompetent mice

“…PSAD + RCA + PCR increases the sensitivity and specificity of HBV cccDNA detection compared to Southern blotting [ 19 ]. The products were separated by 2% agarose gel electrophoresis and visualized under UV light ( Fig 3B ).…”

“…Reactions were performed at 30°C for 16 h and terminated at 65°C for 10 min. Finally, the production of RCA was identified by PCR using a pair of cccDNA-selective labeled primers that target the gap region between the two direct repeat regions of the viral genome [ 19 , 20 ].…”

Detection of HBV Covalently Closed Circular DNA

“…The methods described above are for the extraction and enrichment of cccDNA. However, in situ cccDNA analysis in formalin-fixed paraffin-embedded (FFPE) liver tissues requires different procedures that will be described in the section on cccDNA detection and quantification [ 21 , 28 ]. Before further detection of cccDNA, nucleases such as plasmid-safe adenosine triphosphate (ATP)-dependent deoxyribonuclease (PSAD), mung bean DNase or T5 exonuclease are often used to degrade contaminating non-cccDNA forms [ 20 , 21 , 28 , 30 , 31 , 38 , 39 , 54 , 63 , 69 ], while there are different studies showing that PSAD is not able to clear all rcDNA [ 56 , 67 ].…”

“…The widely accepted method for cccDNA detection is Southern blotting, which is insensitive, complex, time-consuming and not suitable for high-throughput drug screening. To resolve this problem and facilitate research on cccDNA, many new methodologies, including polymerase chain reaction (PCR)-based methods, Invader assays, in situ hybridization, and surrogates, have recently been applied to detect and quantify cccDNA [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ]. In this review, we summarize and compare currently available approaches for cccDNA detection.…”