Application Of High-sensitivity Digital PCR In The Detection Of HBV CccDNA

Objectives:Hepatitis B virus(HBV)covalently closed circular DNA(cccDNA)is a key factor in maintaining viral replication.HBV is difficult to be cleared after infecting liver cells.Long-term infection may lead to chronic hepatitis B(CHB),cirrhosis and hepatocellular carcinoma(HCC).Therefore,in order to monitor the level of intrahepatic cccDNA of CHB patients and evaluate the efficacy of antiviral drugs during clinical antiviral therapy,there is an urgent need for accurate and reliable detection of cccDNA methods.However,the content of cccDNA in liver cells is extra lower and its structure is difficult to distinguish from HBV relaxed circular DNA(rcDNA),which hinders the development of quantitative detection of cccDNA in the liver during antiviral therapy.In the current methods for detecting cccDNA,Southern Blot is regarded as the gold standard,and rolling circle amplification(RCA)has high specificity,but these two methods cannot detect cccDNA quantitatively.Quantitative PCR(qPCR)can detect cccDNA quantitatively,but it relies on standards curves and is not sensitive enough to detect low-copy cccDNA.Due to its technical reasons,droplet digital PCR(ddPCR)can be used for absolute quantitative detection of target molecules without relying on standard products,thus overcoming the disadvantages of other traditional PCR,such as relying on standard products and failing to detect single-copy genes.ddPCR can meet the requirements of high sensitivity,high accuracy,and high-throughput screening required in clinical testing.Therefore,this study aims to use high-sensitivity digital PCR,combined with T5 exonuclease(T5 Exo)treatment and uracil DNA glycosylase(UNG)anti-pollution system to establish a highly sensitive ddPCR method for detecting cccDNA.Methods:(1)Detection of sensitivity and accuracy of ddPCR:The HBV supercoiled plasmid that simulates the cccDNA structure is diluted in a gradient as the amplification template,and detected by ddPCR to analyze the correlation between the theoretical value and the actual value of the cccDNA detection method.Furthermore,a series of low-copy gradient plasmid DNA were detected by ddPCR and qPCR respectively,and the limit of detection(LOD)values of ddPCR and qPCR were calculated and analyzed respectively,and the sensitivity of ddPCR and qPCR for detecting cccDNA was compared.(2)Optimization of UNG in the ddPCR application system:UNG is added to the ddPCR reaction system,and the optimal concentration is established by detecting the influence of different concentrations of UNG on the generation of ddPCR droplets.(3)Comparison of the effects of different nuclease treatments on the detection of cccDNA:the replication intermediates of virus particles in HepAD38 cells including single-stranded DNA(ssDNA)and rcDNA,and cccDNA in the nucleus are extracted separately.Plasmid-safe ATP-dependent DNase(PSAD),exonucleaseⅢ(ExoⅢ)and T5 Exo were used to digest these replication intermediates or cccDNA,respectively.Finally,Southern Blot and ddPCR were used to analyze ability of PSAD,ExoⅢand T5 Exo to digest and degrade ss DNA and rc DNA.(4)The digestion efficiency of the three enzymes in quantitative detection of cccDNA in liver cancer cells was evaluated by ddPCR:agarose gel electrophoresis and ddPCR were used to analyze the degradation ability of PSAD,ExoⅢand T5 Exo on genomic DNA from liver cancer cells.(5)The cccDNA in 26 pairs of matched HBV-infected liver cancer tissues was detected by ddPCR:According to the above results,using the method established in clinical samples to detect the level of cccDNA,and then the difference in cccDNA content between liver cancer tissues and adjacent tissues were analyzed.Results:(1)Detection of sensitivity and accuracy of ddPCR:HBV supercoiled DNA was used as a template and tested by ddPCR.The results showed that when the cccDNA concentration was between 6.5 copies and 65000copies,the theoretical value of ddPCR had a good correlation with the actual value(10~0-10~5copies/reaction).The LOD value of ddPCR is 5.918copies/reaction,and the LOD value of qPCR is 54.854 copies/reaction,indicating that the sensitivity of ddPCR is higher than that of qPCR.(2)Optimization of UNG in the ddPCR application system:When the concentration of UNG enzyme is less than 0.3U,it has no obvious effect on the generation of droplets in the ddPCR system.After 0.2U UNG is used to process the sample,the contaminated DNA in the system has been degraded to the maximum.(3)Comparison of the effects of different nuclease treatments on the detection of cccDNA:Southern Blot and ddPCR tests showed that T5 Exo has the strongest ability to degrade rcDNA,dslDNA and ssDNA among HBV replication intermediates extracted from HepAD38 cells.ddPCR test showed that T5 Exo has the strongest ability to digest rcDNA in nuclear DNA extracted from HepAD38 cells.(4)The digestion efficiency of the three enzymes in quantitative detection of cccDNA in liver cancer cells was evaluated by ddPCR:Agarose gel electrophoresis and ddPCR detection showed that T5 Exo has the strongest ability to digest and degrade genomic DNA among DNA extracted from liver cancer cells.(5)The cccDNA in 26 pairs of matched HBV-infected liver cancer tissues was detected by ddPCR:The distribution of cccDNA in the liver is 1-10 copies/10~4 cells.The copy number of cccDNA in liver cancer tissue(median:2.60 copy/10~4cells)is significantly lower than the copy number of cccDNA in adjacent tissues(median:7.07 copy/10~4 cells).