Abstract
Chronic hepatitis B virus (HBV) infection affects approximately 240 million people worldwide and remains a serious public health concern because its complete cure is impossible with current treatments. Covalently closed circular DNA (cccDNA) in the nucleus of infected cells cannot be eliminated by present therapeutics and may result in persistence and relapse. Drug development targeting cccDNA formation and maintenance is hindered by the lack of efficient cccDNA models and reliable cccDNA detection methods. Southern blotting is regarded as the gold standard for quantitative cccDNA detection, but it is complicated and not suitable for high-throughput drug screening, so more sensitive and simple methods, including polymerase chain reaction (PCR)-based methods, Invader assays, in situ hybridization and surrogates, have been developed for cccDNA detection. However, most methods are not reliable enough, and there are no unified standards for these approaches. This review will summarize available methods for cccDNA detection. It is hoped that more robust methods for cccDNA monitoring will be developed and that standard operation procedures for routine cccDNA detection in scientific research and clinical monitoring will be established.
Highlights
Chronic hepatitis B virus (HBV) infection increases the risk of developing liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC), and it is a leading cause of mortality around the world
PF-relaxed circular DNA (rcDNA) and double-stranded linear DNA (dslDNA) into single-stranded DNA, whereas the closed circular DNA (cccDNA) stays undenatured and its electrophoretic mobility remains unchanged. When it is further digested with EcoRI after denaturation, cccDNA is linearized to genome-length dslDNA, which appears at the 3.2 kb position
Conclusions and Discussion cccDNA plays a key role in chronic hepatitis B virus infection, viral reactivation after drug withdrawal and drug resistance
Summary
Chronic hepatitis B virus (HBV) infection increases the risk of developing liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC), and it is a leading cause of mortality around the world. 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]. We summarize and compare currently available approaches for cccDNA detection
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