Role of hepatitis B virus in development of hepatocellular carcinoma: Focus on covalently closed circular DNA

Table 1 Methods to detect covalently closed circular DNA

Method	Advantages	Disadvantages	Ref.
Blotting
Southern blot	Specific detection of DNA sequences; Able to distinguish cccDNA from other viral DNA species; Reliable and reproducible	Complicated, time-consuming, and costly	[101-102]
PCR-based methods
Conventional qPCR	Simple, accurate, and sensitive; Suitable for high-throughput screening; Rapid and economical	Specificity is reduced if high concentration of rcDNA is present	[100,102]
Semi-nested and nested qPCR	Sensitive and specific; Allows for cccDNA quantification		[100,102,104,106]
Competitive qPCR	Sensitive; Can readily distinguish cccDNA from rcDNA; Allows for cccDNA quantification		[100,102,107]
Droplet-digital PCR	Very sensitive and accurate; Can detect a single copy of cccDNA precisely; Allows for cccDNA quantification		[102,108-114]
Rolling circle amplification qPCR	Very sensitive; cccDNA is visible at single-cell resolution	Effective amplification may be hindered by cross-linked proteins or diffusion of DNA	[100,102,115-117]
Magnetic capture hybridization PCR	Sensitive and specific; Allows for selective isolation of cccDNA; Reproducible	Unable to capture all cccDNA completely; Complicated and costly	[100,102,118-119]
Invader Assay	Specific, simple, and reproducible		[100,102,121-124]
In situ hybridization	Specific; Can distinguish different types of nucleic acids; Visible at single-cell resolution	Complicated probe design	[100,102,125-126]
Indirect method
Surrogate markers	Non-invasive; Convenient and cost-effective; Suitable for high-throughput screening	Indirect	[100,114,126-128,131-135]

cccDNA: Covalently closed circular DNA.