Detection of hepatitis B virus covalently closed circular DNA in paraffin-embedded and cryo-preserved liver biopsies of chronic hepatitis B patients

Abstract

Recovery of hepatocytes and cccDNA in FFPE tissue was lower, but intrahepatic cccDNA in FFPE biopsies were comparable with cryo-preserved liver tissue. Therefore, FFPE liver tissue is an attractive alternative for cccDNA analysis when cryo-preserved tissue is not available.

Detection of HBV Covalently Closed Circular DNA

“…Some reports have shown that cccDNA is present in patients’ sera and that it is a marker of off-treatment virological relapse [11,26,32,34,114], whereas others have refuted the existence of cccDNA in serum and have used DNA extracted from CHB patients’ sera as a negative control in the quantification of intracellular cccDNA [39,42,63]. Do some non-liver cells, such as PBMCs, support HBV infection and formation of cccDNA?…”

“…Lysis buffer containing sodium dodecyl sulfate (SDS), proteinase K or guanidine thiocyanate, 2-mercaptoethanol and 2% Sarkosy is often used for this purpose, followed by centrifugation to discard the cell debris. The supernatant is further extracted with phenol chloroform and precipitated with ethanol [20,32,33,42,45,65,66]. The QIAamp ® DNA blood & tissue Mini Kit (QIAGEN, Hilden, Germany) and NucleoSpin ® Tissue kit (Macherey-Nagel, Düren, Germany) have also been used to extract total DNA in many reports [26,30,31,35,38,39,40,41,44,67,68,69]; however, the HBV DNA species and ratios recovered from the QIAGEN and NucleoSpin ® Tissue kit have never been validated by Southern blot and compared to the conventional total DNA method.…”

“…To further increase the sensitiveness and accuracy, Singh et al applied two hybridization fluorescence resonance energy transfer (FRET) probes in LightCycler TM (Roche Diagnostic, Mannheim, Germany) real-time PCR [41] which were able to maintain a cccDNA:rcDNA specificity ratio greater than 1:10,000. Due to the inherent speed, simplicity and sensitivity of qPCR, subsequent efforts to optimize general real-time PCR conditions [11,12,13,26,32,33,34,38,54,63,69,85,86,87,88,89] (including primers, probe design and sample handling) to effectively distinguish cccDNA from other HBV DNA forms have been made without sacrificing the high amplification efficiency and sensitivity of the method.…”

“…However, the quantity of cccDNA in FFPE liver tissue is 100-fold lower than that in cryo-preserved liver tissue [32], such that the sensitivity of regular qPCR cannot meet pathologists’ needs. Zhong et al combined rolling circle amplification (RCA) with real-time PCR to address this problem [30,105].…”

“…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.…”