Patent Publication Number: US-9840742-B2

Title: Detection of hepatitis B virus (HBV) DNA and methylated HBV DNA in urine of patients with HBV-associated hepatocellular carcinoma

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application claims priority to U.S. Provisional Patent Application No. 62/012,618, filed Jun. 16, 2014, the contents of which are hereby incorporated by reference in its entirety. 
    
    
     GOVERNMENT FUNDING 
     This invention was (made with government support under 2R44CA165312-02, awarded by the National Cancer Institute. The government has certain rights in the invention. 
    
    
     REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY 
     The content of the electronically submitted sequence listing, file name JBS0527720010.txt, size 8,560 bytes; and date of creation Aug. 21, 2015, filed herewith, is incorporated herein by reference in its entirety. 
     TECHNICAL FIELD 
     This invention relates to a method for detecting and quantitatively measuring hepatitis B virus (HBV) DNA, and its modifications including DNA methylation, and the modification in nucleic acid sequences associated with cancer using samples from tissue and biological fluid (blood or urine) of patients with chronic HBV infection. 
     BACKGROUND 
     Cancer is a disease of the genome. Identification of the genetic and epigenetic changes, such as aberrant DNA mutations and modifications underlying the development of carcinogenesis should provide unambiguous detection of cancer. These molecular changes, in concert with the genetic makeup of a patient, determine the clinical phenotype of the tumor, its response to a given treatment, and the patient&#39;s prognosis. 
     Hepatocellular carcinoma (HCC) is the most frequent cancer in certain parts of the world, and the fifth most cancer common worldwide. Increased incidence of HCC is associated with infections of hepatitis B virus and hepatitis C virus (El-Serag &amp; Mason, 1999). Surgical resection is often curative if the disease is localized and diagnosis occurs early (Klintmalm, 1998). However, diagnosis of HCC is often late, resulting in extensive liver impairment associates with a poor prognosis, often due to late diagnosis. Late diagnosis of HCC is due to limited indicators. For example, among patients with underlying cirrhotic disease, a progressive increase in alpha-fetoprotein (AFP) and/or in alkaline phosphatase or a rapid deterioration of hepatic function may be the only clue to the presence of cancer. 
     Despite the availability of a preventative vaccine, chronic HBV infection remains a global health issue affecting more than 3500 million cases of HBV-related HCC (HBV-HCC). The HBV genome has 2-3 typical CpG islands depending on the genotype (Vivekanandan, Thomas, &amp; Torbenson, 2008; Y. Zhang et al., 2013). Interestingly, these CpG islands are located at strategic locations in the regulatory elements of the HBV genome. For example, CpG island 1 is located in the first exon start site for the S (surface antigen) gene, and CpG islands 2 and 3 cover the enhancer II and the promoter of pregenomic RNA and the first exon start site of the polymerase gene, respectively (Moolla, Kew, &amp; Arbuthnot, 2002; Vivekanandan et al., 2008). Although the virion DNA was found to be mostly unmethylated in both tissue culture and patient serum (Kaur et al., 2010; Vivekanandan et al., 2008), DNA methylation of the intranuclear HBV genome has been associated with repression of gene transcription in cultures (Guo, Li, Mu, Zhang, &amp; Yan, 2009; Kim et al., 2011; Zhang, Hou, &amp; Lu, 2013). Despite the difficulty of dissecting HBV DNA in diseased tissues, higher levels of methylation of CpG islands were found in HCC tissues compared to hepatitis and cirrhosis tissues, thus, methylation of HBV DNA has been associated with hepatocarcinogenesis. 
     Current methods of detection of HBV DNA genome and methylated HBV DNA are either not available or insensitive for fragmented DNA such as formalin fixed paraffin embedded (FFPE) tissues, DNA isolated from body fluids, merely qualitative. There remains a need for assays to quantitatively measure for the HBV DNA and its modifications (methylation) that can be used to monitor for the presence of HBV DNA and its modifications that are related to HCC and to screen for HCC so that HCC can be detected early and administered early. 
     SUMMARY 
     One aspect of the present application is a method of detecting the presence or absence of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) in a mammal, comprising:
         (i) isolating DNA from one or more body fluids or tissues from said mammal;   (ii) quantifying HBV DNA by real-time quantitative PCR assays and the methylation level of one or more regions of HBV DNA with a quantitative methylation assay;   (iii) comparing the level of methylation of the one or more regions of DNA with the level of methylation of the one or more regions of DNA in one or more control body fluids or tissues from mammals known not to have HCC; and   (iv) detecting the presence or absence of HCC, with elevated methylation levels in the one or more regions of DNA of the mammals as compared to the level of methylation in the one or more regions of DNA in the one or more control body fluids or tissues indicating the presence of the cancer, and the absence of elevated methylation levels indicating the absence of HCC.       

     In one embodiment, the body fluids are from circulation body fluids. 
     In another embodiment, the body fluids is selected from the group consisting of: (i) urine; (ii) blood; (iii) serum; (iv) plasma; and (v) salvia. 
     In yet another embodiment, the mammal is a human being. 
     In yet another embodiment, the one or more regions of DNA are the CpG islands 1, 2 or 3 of the HBV genome, and the aberrant methylation of the CpG islands 1, 2 or 3, as determined by qMSP, indicates HCC. 
     In one embodiment, the quantitative methylation assay comprises a quantitative bisulfite specific PCR (BSP) to quantify bisulfite-treated HBV DNA and a quantitative methylation specific PCR (MSP). 
     In another embodiment, the one or more regions of HBV DNA is CpG island 1, wherein the forward primer, reverse primer and the probe of the BSP are nucleotide sequences of SEQ ID No. 1, SEQ ID No. 2, SEQ No. 3, respectively, wherein the forward primer, reverse primer and the probe of the MSP are nucleotide sequences of SEQ ID No. 4, SEQ ID No. 5, SEQ No. 6, respectively, wherein the HBV DNA is quantified by real-time quantitative PCR using a forward primer, a reverse primer and a probe, wherein the forward primer, the reverse primer and the probe of the real-time quantitative PCR are nucleotide sequences of SEQ ID No. 25, SEQ ID No. 26 and SEQ ID No. 27, respectively. 
     In another embodiment, the one or more regions of HBV DNA is CpG island 2, wherein the forward primer, reverse primer and the probe of the BSP are nucleotide sequences of SEQ ID No. 7, SEQ ID No. 8, SEQ No. 9, respectively, wherein the forward primer, reverse primer of the MSP are the nucleotide sequences of SEQ ID No. 10 and SEQ ID No. 11, respectively, wherein the HBV DNA is quantified by real-time quantitative PCR using a forward primer, a reverse primer and a probe, wherein the forward primer and the reverse primer of the real-time quantitative PCR are nucleotide sequences selected from the group consisting of: (i) SEQ ID No. 28 and SEQ ID No. 29, respectively; (ii) SEQ ID No. 30 and SEQ ID No. 31, respectively; (iii) SEQ ID No. 32 and SEQ ID No. 33, respectively; (iv) SEQ ID No. 34 and SEQ ID No. 35, respectively; (v) SEQ ID No. 36 and SEQ ID No. 37, respectively. 
     In another embodiment, the one or more regions of HBV DNA is CpG island 3, wherein the forward primer, reverse primer and the probe of the BSP are nucleotide sequences of SEQ ID No. 12, SEQ ID No. 13, SEQ No. 14, respectively, wherein the forward primer, reverse primer and the probe of the MSP are the nucleotide sequences of SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No. 17, respectively, wherein the HBV DNA is quantified by real-time quantitative PCR using a forward primer, a reverse primer and a probe, wherein the forward primer, the reverse primer and the probe of the real-time quantitative PCR are nucleotide sequences of SEQ ID No. 38, SEQ ID No. 39 and SEQ ID No. 40, respectively. 
     In another embodiment, the MSP is a two-step MSP and the one or more regions of HBV DNA is CpG island 3, wherein the forward primer, reverse primer and the probe of the BSP are nucleotide sequences of SEQ ID No. 12, SEQ ID No. 13, SEQ No. 14, respectively, wherein the forward primer, reverse primer of the first step MSP are nucleotide sequences of SEQ ID No. 18 and SEQ ID No. 16, respectively, wherein the forward primer, reverse primer of the second step MSP are nucleotide sequences of SEQ ID No. 19 and SEQ ID No. 20, respectively, wherein the HBV DNA is quantified by real-time quantitative PCR using a forward primer, a reverse primer and a probe, wherein the forward primer, the reverse primer and the probe of the real-time quantitative PCR are nucleotide sequences of SEQ ID No. 38, SEQ ID No. 39 and SEQ ID No. 40, respectively. 
     In another embodiment, the MSP is a two-step MSP and the one or more regions of HBV DNA is CpG island 3, wherein the forward primer, reverse primer and the probe of the BSP are nucleotide sequences of SEQ ID No. 12, SEQ ID No. 13, SEQ No. 14, respectively, wherein the forward primer, reverse primer of the first step MSP are nucleotide sequences of SEQ ID No. 21 and SEQ ID No. 22, respectively, wherein the forward primer, reverse primer and the probe of the second step MSP are nucleotide sequences of SEQ ID No. 21, SEQ ID No. 23 and SEQ ID No. 24, respectively, wherein the HBV DNA is quantified by real-time quantitative PCR using a forward primer, a reverse primer and a probe, wherein the forward primer, the reverse primer and the probe of the real-time quantitative PCR are nucleotide sequences of SEQ ID No. 38, SEQ ID No. 39 and SEQ ID No. 40, respectively. 
     Another aspect of the present application is a kit for detecting the presence or absence of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) in a mammal, comprising:
         (i) A forward primer and a reverse primer for a quantitative bisulfite specific PCR (BSP) to quantify bisulfite-treated HBV DNA;   (ii) A forward primer and a reverse primer for a quantitative methylation specific PCR (MSP); and   (iii) A forward primer and a reverse primer to quantify HBV DNA by real-time quantitative PCR.       

     In one embodiment, the kit further comprises a probe for BSP, a probe for MSP and a probe for he real-time quantitative PCR, wherein the forward primer, the reverse primer and the probe for BSP are nucleotide sequences of SEQ ID No. 1, SEQ ID No. 2, SEQ No. 3, respectively, wherein the forward primer, reverse primer and the probe of the MSP are nucleotide sequences of SEQ ID No. 4, SEQ ID No. 5, SEQ No. 6, respectively, wherein the forward primer, reverse primer and the probe for the real-time quantitative PCR are nucleotide sequences of SEQ ID No. 25, SEQ ID No. 26 and SEQ No. 27, respectively. 
     In another embodiment, the kit further comprises a probe for BSP, wherein the forward primer, the reverse primer and the probe for BSP are nucleotide sequences of SEQ ID No. 7, SEQ ID No. 8, SEQ No. 9, respectively, wherein the forward primer and the reverse primer for MSP are nucleotide sequences of SEQ ID No. 10 and SEQ ID No. 11, respectively, wherein the forward primer and the reverse primer for the real-time quantitative PCR are nucleotide sequences selected from the group consisting of: (i) SEQ ID No. 28 and SEQ ID No. 29, respectively; (ii) SEQ ID No. 30 and SEQ ID No. 31, respectively; (iii) SEQ ID No. 32 and SEQ ID No. 33, respectively; (iv) SEQ ID No. 34 and SEQ ID No. 35, respectively; (v) SEQ ID No. 36 and SEQ ID No. 37, respectively. 
     In another embodiment, the kit further comprises a probe for BSP, a probe for MSP and a probe for he real-time quantitative PCR, wherein the forward primer, the reverse primer and the probe for BSP are nucleotide sequences of SEQ ID No. 12, SEQ ID No. 13, SEQ No. 14, respectively, wherein the forward primer, the reverse primer and the probe for MSP are nucleotide sequences of SEQ ID No. 15, SEQ ID No. 16, SEQ No. 17, respectively, wherein the forward primer, the reverse primer and the probe for the real-time quantitative PCR are nucleotide sequences of SEQ ID No. 38, SEQ ID No. 39 and SEQ No. 40, respectively. 
     In another embodiment, the kit further comprises a probe for BSP and a probe for he real-time quantitative PCR, wherein the MSP is a two-step MSP, wherein the forward primer, reverse primer and the probe of the BSP are nucleotide sequences of SEQ ID No. 12, SEQ ID No. 13, SEQ No. 14, respectively, wherein the forward primer, reverse primer of the first step MSP are nucleotide sequences of SEQ ID No. 18 and SEQ ID No. 16, respectively, wherein the forward primer, reverse primer of the second step MSP are nucleotide sequences of SEQ ID No. 19 and SEQ ID No. 20, respectively, wherein the forward primer, the reverse primer and the probe for the real-time quantitative PCR are nucleotide sequences of SEQ ID No. 38, SEQ ID No. 39 and SEQ No. 40, respectively. 
     In another embodiment, the kit further comprises a probe for BSP and a probe for he real-time quantitative PCR, wherein the MSP is a two-step MSP, wherein the forward primer, reverse primer and the probe of the BSP are nucleotide sequences of SEQ ID No. 12, SEQ ID No. 13, SEQ No. 14, respectively, wherein the forward primer, reverse primer of the first step MSP are nucleotide sequences of SEQ ID No. 21 and SEQ ID No. 22, respectively, wherein second step MSP further comprising a probe, wherein the forward primer, reverse primer and the probe of the second step MSP are nucleotide sequences of SEQ ID No. 21, SEQ ID No. 23 and SEQ ID No. 24, respectively, wherein the forward primer, the reverse primer and the probe for the real-time quantitative PCR are nucleotide sequences of SEQ ID No. 38, SEQ ID No. 39 and SEQ No. 40, respectively. 
     Another aspect of the present application is a method of quantifying HBV DNA, comprising: amplifying and quantifying HBV DNA by real-time quantitative PCR assays using a forward primer and a reverse primer, wherein the forward primer and the reverse primer of the real-time quantitative PCR are nucleotide sequences selected from the group consisting of: (i) SEQ ID No. 25 and SEQ ID No. 26, respectively; (ii) SEQ ID No. 38, SEQ ID No. 39, respectively; (iii) SEQ ID No. 28 and SEQ ID No. 29, respectively; (iv) SEQ ID No. 30 and SEQ ID No. 31, respectively; (v) SEQ ID No. 32 and SEQ ID No. 33, respectively; (vi) SEQ ID No. 34 and SEQ ID No. 35, respectively; (vii) SEQ ID No. 36 and SEQ ID No. 37, respectively. 
     In another embodiment, the forward primer and the reverse primer are the nucleotide sequences of SEQ ID No. 25 and SEQ ID No. 26, respectively, wherein the real-time quantitative PCR assay further utilizes a probe, wherein the probe is nucleotide sequence of SEQ ID No. 27. 
     In another embodiment, the forward primer and the reverse primer are the nucleotide sequences of SEQ ID No. 38 and SEQ ID No. 39, respectively, wherein the real-time quantitative PCR assay further utilizes a probe, wherein the probe is nucleotide sequence of SEQ ID No. 40. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a diagram of the HBV genome (NC_003977.1) indicating the location of the three CpG islands and the primers for PCR assays used in the present application. 
         FIG. 2  is the methylation profile of the HBV genome by bisulfite sequencing of DNA isolated from cell cultures or liver tissues from HBV patients. 
         FIG. 3  shows the quantitative analysis of HBV DNA methylation in infected diseased livers. 
         FIG. 4  shows comparison of HBV and host gene methylation in HBV-HCC. 
         FIG. 5  shows the comparison of the amount of methylated HBV CpG island 3 DNA determined by a short amplicon MSP assay, in urine of patients with HBV-related liver diseases. 
         FIG. 6  shows the detection of methylated CpG 3 DNA by the a short amplicon MSP assay, in urine of patients with HBV infection as a biomarker to distinguish HCC from cirrhosis. 
         FIG. 7  shows the detection of methylated CpG 3 DNA by a short amplicon MSP assay, in urine of patients with HBV infection as a biomarker to distinguish HCC from hepatitis+cirrhosis. 
         FIG. 8  shows results using the protocol for quantification of bisulfite converted CpG island 1 of the HBV genome. 
         FIG. 9  shows results using the protocol for the quantification of methylated CpG island 1 of the HBV genome. 
         FIG. 10  shows results using the protocol for the quantification of bisulfite converted CpG island 2 of the HBV genome. 
         FIG. 11  shows results using the protocol for the quantification of methylated CpG island 2 of the HBV genome. 
         FIG. 12  shows results using the protocol for the quantification of bisulfite converted CpG island 3 of the HBV genome. 
         FIG. 13  shows results using the protocol for the quantification of methylated CpG island 3 of the HBV genome. 
         FIG. 14  shows results using the protocol for detection of circulation derived methylated CpG island 3 of the HBV genome by using HBV-MSP Short Amplicon Assay (mHBV). 
         FIG. 15  shows results using the protocol for quantification of methylated CpG island 3 of the HBV genome by a short amplicon MSP assay that can be used for fragmented DNA. 
         FIG. 16  shows results using the protocol for quantification of genomic CpG island 1 of the HBV genome. 
         FIG. 17  shows results using the protocol for quantification of genomic CpG island 2 of the HBV genome. 
         FIG. 18  shows results using the protocol for quantification of genomic CpG island 2 of the HBV genome. 
         FIG. 19  shows results using the protocol for quantification of genomic CpG island 2 of the HBV genome. 
         FIG. 20  shows results using the protocol for quantification of genomic CpG island 2 of the HBV genome. 
         FIG. 21  shows results using the protocol for quantification of genomic CpG island 2 of the HBV genome. 
         FIG. 22  shows results using the protocol for quantification of genomic CpG island 3 of the HBV genome. 
     
    
    
     DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 
     The present subject matter may be understood more readily by reference to the following detailed description taken in connection with the accompanying examples, which form a part of this disclosure. It is to be understood that this invention is not limited to the specific devices, methods, applications, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention. 
     Also, as used in the specification including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The term “plurality,” as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable. 
     Hepatitis B virus (HBV) is a hepatotropic virus causing hepatitis, cirrhosis and hepatocellular carcinoma (HCC). In patients, HBV DNA circulates in blood as virion DNA and exists in hepatocytes both as nuclear form (episomal cccDNA and integrated DNA) and as cytoplasmic core DNA form. It has been reported that HBV infection up-regulates DNA (cytosine-5) methyltransferases and thus induces epigenetic changes in the host cells. Understanding the methylation status of the HBV DNA in its different forms can potentially provide insight into the pathogenesis of HBV-related liver diseases including hepatocarcinogenesis. Previous studies conducted in patient serum and in cell cultures have demonstrated that the CpG islands of HBV virion DNA are in the unmethylated state, and cccDNA methylation has been extensively studied for its role in the regulation of transcription, but very few studies have investigated the methylation status of the HBV DNA in various disease stages of human liver tissues. 
     The discovery of the association between CpG island 3 methylation and HCC development is illustrated in  FIGS. 3 to 7 .  FIG. 3  shows the methylation profile of CpG island 3 in different liver diseases including hepatitis, cirrhosis, and HCC by bisulfite PCR sequencing, suggesting that the methylation of the CpG island 3 is associated with HCC development; Thus, it can be a potential biomarker for the early detection of HCC and can be used for HCC screening using urine as body fluid as described in  FIGS. 5 to 7 . 
       FIG. 1  is a diagram of the HBV genome (NC_003977.1) indicating the location of the three CpG islands and the primers for PCR assays used in the present application. Black rectangles represent the HBV regions; polymerase, enhancer II, basal core promoter, precore, preS and X gene. These regions correspond to the dashed line representing the HBV genome with vertical gray bars indicating nucleotide location. The three CpG islands are indicated by horizontal grey bars below the dashed line. The CpG site distribution in the HBV genome (solid black line) is depicted by vertical grey lines each representing an individual CpG site. The boxes with black borders on the CpG distribution map indicate the regions targeted by the bisulfite sequencing PCR and are enlarged below for better resolution of the CpG sites. The numbering of the CpG sites in the enlarged regions corresponds to that used in  FIG. 2 . The arrows below this enlarged bisulfite sequencing region indicate primer locations for the specified PCR assays. Detailed primer information is in Table 3.  FIG. 1  shows CpG Islands reports potential CpG island regions using the method described by Gardiner-Garden and Frommer (1987). The calculation is performed using a 200 bp window moving across the sequence at 1 bp intervals. CpG islands are defined as sequence ranges where the Obs/Exp value is greater than 0.6 and the GC content is greater than 50%. 
     As shown in  FIG. 2 , we did not detect methylation in virions. No detectable methylation was identified either in cytoplasmic core, the cccDNA/extra-chromosomal nuclear DNA isolated from HepDE19 cells, or the total DNA from the HepG2.2.15 cell culture. For the integrated DNA, when the DNA was derived from SNU-398 cells, which contain the transcriptionally repressed HBV genome, DNA methylation was detected in all the HBV DNA amplified by BS-PCR primers. When the integrated HBV DNA from Hep3B was analyzed, which is known to express the S antigen, preS and X transcripts, the HBV DNA was mostly unmethylated except for the CpG island 1, which was mostly methylated. 
     As shown in  FIG. 2 , there are regions marked as “data unavailable” such as the CpG island 1 and the 5′ end of the CpG island 2 of the HBV genome in SNU-398 cells, and the middle of the CpG island 2 and CG3 of the HBV genome in Hep3B cells. These were the regions that BS-PCR reactions either failed to generate specific PCR products for DNA sequencing, or the sequencing data were unreadable despite multiple attempts. Many attempts using multiple sets of primers for both untreated and BS-treated DNA were unable to amplify detectable PCR products, perhaps due to the possibility that DNA sequences in these areas might be either deleted during HBV integration, or altered to the extent that none of the tested primers had sufficient homology to prime the PCR reaction. As compared to the reference genome, few nucleotide variants were noted in Hep3B and SNU-398 cells. 
     To obtain the comprehensive methylation profiles for each CpG site of all three known CpG islands in the HBV genomes from both cell cultures and infected tissues, we chose to perform bisulfite PCR (BS-PCR) sequencing on a small set of tissue samples (8 hepatitis, 6 cirrhosis, and 12 HCC) based on the quantity of DNA available to us. The clinicopathological information is summarized in Supplementary Table S1 in Jain et. al., 2015. In order to design the suitable BS-specific primers, we began by performing the genotyping of the HBV DNA in each tissue sample. We then designed BS primers covering three CpG islands in various genotypes of the HBV genome, to amplify the BS-treated HBV DNA from cell cultures and tissue samples ( FIG. 1 , Table 1). 
       FIG. 2  is the Methylation profile of the HBV genome by bisulfite sequencing of DNA isolated from cell cultures or liver tissues from HBV patients. Each box indicates one CpG site (filled, methylated; open, unmethylated; hatch, data unavailable; text, possible sequence variations at CpG site). Top panel shows the results from cell cultures—HepDES19 derived virion, core, cccDNA and total HepG2.215 DNA, followed by DNA isolated from two HBV-integrated HCC cell lines SNU-398 and Hep3B. Subsequent three panels show the results from clinical samples—hepatitis, cirrhosis and HCC aligned to HBV genotype C virus (NC_003977.1). Bottom panel shows results from total tumor tissue DNA (T), cccDNA from tumor (T C ) and adjacent non-HCC tissue (N C ). The genotype of each sample is indicated on the right. 
     We next performed BS-PCR sequencing analysis on the disease tissues. As aforementioned, we developed a total of 24 sets of primers based on genotyping data, 10 sets for the CpG island 1, 12 sets for the CpG island 2, and 2 sets for the CpG island 3. Although we were able to generate specific PCR products from most of the HBV infected tissue DNA to obtain BS sequencing data for most of the CpG sites, there were still regions in the genomes that failed to be amplified, regardless of multiple attempts. Thus, the methylation status was unavailable for those regions, as indicated in the  FIG. 2 . Nevertheless, the information obtained is sufficient for statistical analysis of the methylation of each CpG island comparing HCC tissues to hepatitis and cirrhosis. 
     To compare DNA methylation in different disease groups, we calculated the percentage of CpG sites that were found to contain detectable levels of methylation, which were analyzed for each disease group within each CpG island (Table 1). A low level of methylation was detected in all three CpG islands in the hepatitis samples (6.6%, 6%, and 17.5% in CpG island 1, CpG island 2 and CpG island 3, respectively), whereas, the HBV DNA detected in cirrhosis tissue was mostly unmethylated in CpG island 1 (0%) and CpG island 2 (0.8%), and a low level of methylation was found in CpG island 3 (10.8%). Interestingly, in the HCC samples, although a low percentage of methylation was detected in CpG island 1 and CpG island 2 (16.1% and 8%, respectively), 52.5% of the CpG sites in CpG island 3 were found to be methylated. Furthermore, every CpG site of CpG island 3, except for three, were found to be methylated in 6 of the 12 HCC samples that had methylation. Comparing these three CpG islands within HCC samples, the CpG island 3 is the most methylated (p&lt;0.001, comparing CpG island 3 to CpG island 1 and CpG island 3 to CpG island 2, by Fisher&#39;s two-tailed exact test). When comparing the extent of methylation of CpG island 3 among three disease groups, HCC was significantly higher than that of hepatitis and cirrhosis (p&lt;0.0001, Fisher&#39;s two-tailed exact test, Table 1). Similarly, HCC also had significantly higher levels of methylation in CpG island 1 and CpG island 2, as compared to hepatitis and cirrhosis (CpG island 1, p=0.0007; CpG island 2, p=0.0046, Fisher&#39;s two-tailed exact test, Table 1). There were few nucleotide variants with respect to the reference genome (Genbank # NC 003977.1), as noted in  FIG. 2 , which were mostly in CpG island 2 and the first 2 CpG sites of CpG island 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 HBV DNA methylation in various HBV-infected livers by BS-PCR sequencing. 
               
            
           
           
               
               
            
               
                   
                 CpG island 
               
            
           
           
               
               
               
               
            
               
                 Percent of methylated CpG sites ε   
                 1 
                 2 
                 3 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Hepatitis 
                 6.6%  
                 (5/76) 
                 6.0%  
                 (15/250) 
                 17.5%  
                 (16/91) 
               
               
                 Cirrhosis 
                 0%  
                 (0/60) 
                 0.8%  
                 (2/236) 
                 10.8%  
                 (7/65) 
               
               
                 HCC 
                 16.1%  
                 (20/124) 
                 8.0%  
                 (32/398) 
                 52.5%  
                 (75/143) 
               
            
           
           
               
               
               
               
            
               
                 p value #   
                 0.0007 
                 0.0046 
                 &lt;0.0001 
               
               
                 HCC vs. (Hepatitis and Cirrhosis) 
                   
                   
                   
               
               
                   
               
               
                   ε The percent of methylated CpG sites was calculated as the number of methylated CpG sites detected/total CpG sites with valid BS sequencing data (methylated + unmethylated) shown in FIG. 2. 
               
               
                   # Fisher&#39;s two-tailed exact test. 
               
            
           
         
       
     
       FIG. 3  is the quantitative analysis of HBV DNA methylation in infected diseased livers. Scatter plots of the quantity of methylated HBV DNA detected by qMSP assays for each CpG island. Each data point represents one sample and is the average of two duplicate assays per input of 3000 copies of BS-actin DNA. p values indicate the statistical comparison between HCC and non-HCC (hepatitis and cirrhosis) samples for each CpG island by Mann-Whitney U test. 
     To evaluate whether the extent of DNA methylation was associated with the progression of liver diseases, we developed quantitative MSP (qMSP) assays for each CpG island to measure the DNA methylation in a larger sample size. As some of the HBV infected tissues may not contain any detectable HBV DNA, each DNA sample was first subjected to the quantitative HBV DNA PCR assays for CpG island 1 and CpG island 3, as described in the Materials and Methods. Only samples positive for at least one HBV DNA assay were subjected to methylation analysis. The clinicopathological characteristics of the study subjects (74 HCC, 29 hepatitis and 13 cirrhosis) are listed in Supplementary Table S2 of Jain et. al., 2015. Three qMSP assays were performed to quantify the level of methylation for each of the three CpG islands. As shown in  FIG. 3 , there is no statistically significant difference (p&gt;0.05, Mann Whitney U-test) between the methylation levels of HCC and non-HCC (hepatitis and cirrhosis) tissues in CpG island 1 and CpG island 2. In contrast, methylation of CG3 in HCC tissues (mean=1250.5, standard deviation=3504) was significantly higher than in non-HCC tissue (hepatitis and cirrhosis, mean=174.6, standard deviation=534) tissues (p&lt;0.05, Mann Whitney U-test). Of the 74 HBV-HCC cases, 9 are HCV positive, 45 are HCV negative, and the HCV status of the remaining 20 is unknown. HCV infection is also known to cause aberrant methylation and can be a potential confounding factor. Hence, we compared the methylation levels of CpG island 3 in HBV-positive, HCV-negative HCC (n=45) to HBV-infected hepatitis and cirrhosis samples. The results confirmed that methylation of CpG island 3 in this subset of HCC tissues was still significantly higher than in hepatitis and cirrhosis tissues (p&lt;=0.05, Mann Whitney U-test). We also observed no significant differences in the levels of CpG island 3 methylation in the HBV-HCC samples that were categorized by their HCV status (p=0.695, Kruskal Wallis test). Interestingly, we observed higher levels of methylation in CpG island 1 and CpG island 3 of hepatitis samples as compared to cirrhosis samples, although only methylation of CpG island 1 was significantly higher in hepatitis samples as compared to cirrhosis samples (p=0.05, Mann Whitney U test). No methylation was observed in either hepatitis or cirrhosis samples in CpG island 2. 
       FIG. 4  shows comparison of HBV and host gene methylation in HBV-HCC. More specifically, it shows distribution of HBV and host genome methylation categorized by their serum alfa-fetoprotein (AFP) levels in HBV-HCC samples (n=68). Each column represents one sample. In the AFP row, filled rectangles indicate serum AFP greater than 20 ng/ml while open rectangles indicate a serum AFP level less than 20 ng/ml. Filled rectangles in mCpG1, mCpG2, mCpG3, mAPC, mGSTP1 and mRASSF1A indicate methylated status while open rectangles indicate no detectable methylation. 
     It has been shown that HBV infection upregulates DNA methyltransferase activity, which leads to simultaneous methylation of HBV DNA and host CpG islands in cell culture experiments. DNA methylation of many tumor suppressor genes has been associated with carcinogenesis. Among these known tumor-associated, aberrant-methylation events, methylation of the APC, GSTP-1, and RASSF1A genes were found to be associated with HCC. It is therefore of interest to investigate whether the HBV DNA methylation correlates with these three HCC-associated host gene methylation events. BS-treated HCC DNA was subjected to previously developed quantitative MSP assays for these three genes ( FIG. 4 ). The Spearman&#39;s rho test was used to determine the correlation co-efficiency (Table 2). When comparing methylation of genes within the host genome, there is a significant correlation (p&lt;0.01) between either of the two host genes, and within the HBV genome in HBV-related HCC, there is also a significant correlation (p&lt;0.01) between CpG island 1 and CpG island 3 of the HBV genome in HCC. The only significant correlation between HBV DNA and host genes was found between methylation of CpG island 3 and RASSF1A. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Correlation of DNA methylation of the HBV genome with three  
               
               
                 known HCC associated host genes in HCC tissue. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 mCpG1 
                 mCpG3 
                 mAPC 
                 mGSTP1 
               
               
                 Genes 
                 Statistic 
                 (n = 74) 
                 (n= 74) 
                 (n = 71) 
                 (n = 71) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 mCpG3 
                 Spearman&#39;s  
                 .471 
                   
                   
                   
               
               
                 (n = 74) 
                 rho 
                   
                   
                   
                   
               
               
                   
                 p value 
                 &lt;0.001 
                   
                   
                   
               
               
                 mAPC 
                 Spearman&#39;s  
                 0.098 
                 0.094 
                   
                   
               
               
                 (n = 71) 
                 rho 
                   
                   
                   
                   
               
               
                   
                 p value 
                 0.418 
                 0.435 
                   
                   
               
               
                 mGSTP1 
                 Spearman&#39;s  
                 0.233 
                 −0.008 
                 .324 
                   
               
               
                 (n = 71) 
                 rho 
                   
                   
                   
                   
               
               
                   
                 p value 
                 0.051 
                 0.947 
                 0.006 
                   
               
               
                 mRASSF1A  
                 Spearman&#39;s  
                 0.132 
                 .269 
                 .510 
                 .513 
               
               
                 (n = 71) 
                 rho 
                   
                   
                   
                   
               
               
                   
                 p value 
                 0.271 
                 0.023 
                 &lt;0.001 
                 &lt;0.001 
               
               
                   
               
            
           
         
       
     
     Cell-free circulating DNA has been shown to reflect characteristics of tumor DNA (Chan et al., 2008; Diehl et al., 2008; Forshew et al., 2012; Pathak, Bhutani, Kumar, Mohan, &amp; Guleria, 2006; Stroun, Anker, Lyautey, Lederrey, &amp; Maurice, 1987; Stroun et al., 1989; Tsutsui et al., 2010; Utting, Werner, Dahse, Schubert, &amp; Junker, 2002; Wong, Zhang, Lai, Lau, &amp; Lo, 2003; Wu et al., 2002; Yen et al., 2009; Ziegler, Zangemeister-Wittke, &amp; Stahel, 2002) and are mostly fragmented in 1-2 nucleosomal sizes in some body fluids such as plasma and urine. Attempts have been made to detect cancer genetically in blood, but developing a test of sufficient sensitivity has been challenging (Chan et al., 2008; Kirk et al., 2005; Lleonart et al., 2005; Tsutsui et al., 2010; Wong et al., 2003). Thus, technologies must be developed that would be capable of isolating circulating DNA in quantities sufficient for the analysis of biomarkers and of detecting circulating DNA markers with a sensitivity and specificity sufficient to translate the discovered biomarkers for application in HCC screening. 
     Urine has been used as a source of reporter molecules for urinary tract diseases with great clinical benefit. Urine-based tests are non-invasive and very patient-friendly. The use of urine as a biological fluid for cancer detection is now possible with advances in molecular biomarker assays and recent findings that tumor-derived DNA in circulation can be detected in urine as low-molecular-weight (LMW) urine DNA, which is less than 300 bp in size (Su et al., 2004). A short amplicon MSP assay was developed (as described in the paragraph 61) to quantify the methylated HBV CpG island 3 DNA in urine and other body fluids.  FIG. 5  shows the amount of methylated HBV CpG island 3 DNA determined by a short amplicon MSP assay (as described in the paragraph 61) in urine of patients with HBV-related liver diseases, compared with the amount of methylated HBV CpG island 3 in normal non-cancer subjects. 
       FIG. 6  shows the detection of methylated CpG 3 DNA by a short amplicon MSP assay (as described in the paragraph 61), in urine of patients with HBV infection as a biomarker to distinguish HCC from cirrhosis. The ROC curve was constructed and area under the curve (AUROC) was 0.867. In this small cohort, the AUROC of serum AFP was 0.798. 
       FIG. 7  shows the performance of methylated CpG 3 DNA as a DNA biomarker to distinguish HCC from non-HCC (hepatitis+cirrhosis) in urine of patients with HBV infection, assayed by a short amplicon MSP assay (as described in the paragraph 61). The ROC curve was constructed and AUROC was 0.923 to distinguish HCC from non-HCC in urine. 
     The term “nucleotide amplification reaction” refers to any suitable procedure that amplifies a specific region of polynucleotides (target) using primers. See generally Kwoh et al., Am. Biotechnol. Lab. 8:14 (1990; Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173-1177 (1989); Lizardi et al., BioTechnology 6:1197-1202 (1988); Malek et al., Methods Mol. Biol., 28:253-260 (1994); and Sambrook et al., “Molecular Cloning: A laboratory Manual” (1989)). 
     A “detectable label” is a molecule or atom which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties. 
     The term “effective amount,” in the context of treatment of a disease or disorder refers to the amount of such molecule that is sufficient to inhibit the occurrence or ameliorate one or more clinical or diagnostic symptoms of the disease or disorder in a subject. The term “effective regime” refers to a combination of amount of the agent being administered and dosage frequency adequate to accomplish treatment or prevention of the disease or disorder. 
     Due to the imprecision of standard analytical methods, molecular weights and lengths of polymers are understood to be approximate values. When such a value is expressed as “about” X or “approximately” X, the stated value of X will be understood to be accurate to ±10%. are provided to assist in a further understanding of the inventions. Particular materials used, protocols and conditions are intended to be further illustrative of the inventions and should not be construed to limit the reasonable scope thereof. 
     Provided herein is a suitable method for detecting the presence or absence of a cancer in an individual by determining the level of DNA mutations and aberrant DNA methylation from the individual, comparing the level of DNA mutations and aberrant DNA methylation with a baseline level of DNA mutations and methylation found in one or more control samples from individuals known not to have the cancer, and correlating a finding of elevated methylation and DNA mutations in the individual with an enhanced likelihood that the individual has cancer. The cancer can be hepatocellular carcinoma (HCC) and the control can be non-HCC sample. The examples of genetic DNA mutations (listed in Table 3) and aberrant DNA methylation markers (listed in Table 3) that can be used for HCC screening using urine as body fluid. 
     Provided herein is a suitable method for detecting the presence or absence of HBV DNA in liver tissues or body fluids and whether the HBV DNA is methylated. The examples of oliogs used in each assay for detecting HBV DNA or bisulfite converted HBV DNA with the location of the oligos and conditions of the respective PCR reactions (listed in Table 3). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Primer/Probe sequences and reaction parameters for quantitative PCR assays 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 HBV nt. 
                   
                   
                   
                   
                   
               
               
                 Location 
                 (NC_003977.1) 
                 Assay 
                 Forward 
                 Reverse 
                 Probe/Detection format 
                 PCR conditions 
               
               
                   
               
               
                 CpG Island 1 
                 207-285 
                 BSP 
                 HBV_BSC1F1: 
                 HBV_BSC1R3: 
                 BSC1TQ:[6FAM]TTATAA 
                 95° C. 5 min, (95° C. 
               
               
                   
                   
                   
                 GGTTTTTTTTGTTGAT 
                 CCCCCTAAAAA 
                 TATTATAGAGTTTAGA 
                 10 s 50° C. 15 s, 72° C. 10 s) × 
               
               
                   
                   
                   
                 AAGAATTT (SEQ ID 
                 ATTAAAAAAA 
                 TTYGTGGTGGA[BHQ1] 
                 50 cycles 
               
               
                   
                   
                   
                 NO: 1) 
                 A (SEQ ID NO: 2) 
                 (SEQ ID NO: 3) 
                   
               
               
                   
                 293-402 
                 MSP 
                 HBV_C1F2B: 
                 HBV_C1R5B: 
                 C1MSTQ:[6FAM] 
                 95° C. 5 min, (95° C. 10 s 55° C. 30 s, 
               
               
                   
                   
                   
                 ACGTGTTTTGGTTAA 
                 AATATAATAAA 
                 GTTTTTTAATTTGTTTT 
                 72° C. 10 s) × 50 cycles 
               
               
                   
                   
                   
                 AATTCGTAGTTTTTA 
                 ACGCCGCAAAC 
                 GGTTATCGTTGGATG 
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 4) 
                 ACATC (SEQ ID 
                 [BHQ1] (SEQ ID NO: 6) 
                   
               
               
                   
                   
                   
                   
                 NO: 5) 
                   
                   
               
               
                   
               
               
                 CpG Island 2 
                 1660-1733 
                 BSP 
                 HBV_BSC2F1: 
                 HBV_BSC2R3: 
                 BSC2TQ:[6FAM]ATGTTA 
                 95° C. 5 min, (95° C. 
               
               
                   
                   
                   
                 GGATTTTTGGATTTTT 
                 CCAATCTTTAA 
                 AYGATYGATTTTGAGG 
                 10 s 53° C. 15 s, 72° C. 10 s) × 
               
               
                   
                   
                   
                 AGTAATGTT (SEQ ID 
                 ACAAACAATCT 
                 TATATTTTAA[BHQ1] 
                 50 cycles 
               
               
                   
                   
                   
                 NO: 7) 
                 TTAA (SEQ ID 
                 (SEQ ID NO: 9) 
                   
               
               
                   
                   
                   
                   
                 NO: 8) 
                   
                   
               
               
                   
                 1502-1579 
                 MSP 
                 HBV_C2MF3: 
                 HBV_C2MR3: 
                 SYBR Green 
                 95° C. 5 min, (95° C. 10 s 52° C. 30 s, 
               
               
                   
                   
                   
                 TGTCGTTTCGGTCGAT 
                 CACGATCCGAC 
                   
                 72° C. 10 s) × 45 cycles, melt [95° C. 
               
               
                   
                   
                   
                 TAC (SEQ ID NO: 10) 
                 AAATAAAAA 
                   
                 5 s, 65° C. 60 s, 97° C. 
               
               
                   
                   
                   
                   
                 (SEQ ID NO: 11) 
                   
                 continuous], 40° C. 30 s 
               
               
                   
               
               
                 CpG Island 3 
                 2161-2239 
                 BSP 
                 HBV_BSC3F9: 
                 HBV_BSC3R9.10: 
                 C3BSTQ: 
                 95° C. 10 min, (95° C. 10 s, 52° C. 30 s, 
               
               
                   
                   
                   
                 TTATGTTAATGTTAAT 
                 TTCTCTTCCAA 
                 [6FAM]TTAgATAATTAT 
                 72° C. 10 s) × 50 cycles 
               
               
                   
                   
                   
                 ATGGGTTTAAA (SEQ 
                 AAATAAAACA 
                 Tg+Tgg+T+T+T+TA+TA+ 
                   
               
               
                   
                   
                   
                 ID NO: 12) 
                 A (SEQ ID 
                 T[BBQ] (SEQ ID NO: 14) 
                   
               
               
                   
                   
                   
                   
                 NO: 13) 
                   
                   
               
               
                   
                 2270-2412 
                 MSP 
                 HBV_MSP2F: 
                 HBV_MSP2R: 
                 C3AMSTQ: 
                 95° C. 10 min, (95° C. 10 s, 53° C. 30 s, 
               
               
                   
                   
                   
                 GTGTGGATTCGTATTT 
                 GACGATTAAAA 
                 AACCTACCTCGTCGTCT 
                 72° C. 10 s) × 45 cycles 
               
               
                   
                   
                   
                 TTTTC (SEQ ID NO: 15) 
                 CCTTCGTCT 
                 AACAACAAT (SEQ ID 
                   
               
               
                   
                   
                   
                   
                 (SEQ ID NO: 16) 
                 NO: 17) 
                   
               
               
                   
                 2370-2412 
                 MSP- 
                 HBT_SMF1: 
                 HBV_MSP2R; 
                 N/A 
                 95° C. 5 min, (95° C. 30 s, 50° C. 30 s, 
               
               
                   
                   
                 Step 1 
                 GCTCTTCGTGGTGTG 
                 GACGATTAAAA 
                   
                 72° C. 30 s) × 27 cycles, 72° C. 4 min, 
               
               
                   
                   
                   
                 GTGAAGAAGAATTTT 
                 CCTTCGTCT 
                   
                 4° C. Hold 
               
               
                   
                   
                   
                 TTCGTTTC (SEQ ID NO 
                 (SEQ ID NO: 16) 
                   
                   
               
               
                   
                   
                   
                 18) 
                   
                   
                   
               
               
                   
                   
                 MSP- 
                 HBV_S2F1: 
                 HBV_S2R1: 
                 SYBR Green 
                 95° C. 5 min, (95° C. 10 s, 50° C. 15 s, 
               
               
                   
                   
                 Step 2 
                 TGTGGTGAAGAAGAA 
                 GACGATTAAAA 
                   
                 72° C. 10 s) × 45 cycles, Melting curve, 
               
               
                   
                   
                   
                 TT (SEQ ID NO: 19) 
                 CCTTC (SEQ ID 
                   
                 40° C. 30 s 
               
               
                   
                   
                   
                   
                 NO: 20) 
                   
                   
               
               
                   
                 2375-2430 
                 MSP- 
                 C3.M_F1: 
                 C3.M_R3: 
                 N/A 
                 95° C. 5 min, (95° C. 30 s, 50° C. 30 s, 
               
               
                   
                   
                 Step 1 
                 AGAATTTTTTCGTTTC 
                 AAAATCTTCTA 
                   
                 72° C. 30 s) × 25/40 cycles; 72° C. 4 min, 
               
               
                   
                   
                   
                 GTAGAC (SEQ ID NO: 
                 CGACGCGACG 
                   
                 4° C. hold 
               
               
                   
                   
                   
                 21) 
                 ATTAA (SEQ ID 
                   
                   
               
               
                   
                   
                   
                   
                 NO: 22) 
                   
                   
               
               
                   
                   
                 MSP- 
                 C3.M_F1: 
                 C3.M_R2: 
                 SYBR and Simple Probe 
                 95° C. 5 min, (95° C. 10 s, 53° C. 30 s, 
               
               
                   
                   
                 Step 2 
                 AGAATTTTTTCGTTTC 
                 CTACGACGCGA 
                 FLQ-CG3.MSP SP: 
                 72° C. 10 s) × 45 cycles 
               
               
                   
                   
                   
                 GTAGAC (SEQ ID 
                 CGATTAAAAC 
                 TCGTTTCGTAGACGAA 
                   
               
               
                   
                   
                   
                 NO: 21) 
                 (SEQ ID NO: 23) 
                 GGT--PH (SEQ ID NO: 24) 
                   
               
               
                   
               
               
                 CpG Island 1 
                 246-309 
                 HBV 
                 HBV240F3: 
                 HBVC240R1: 
                 HBV240 TaqMan: 
                 95° C. 5 min, (95° C. 10 s, 54° C. 15 s, 
               
               
                   
                   
                 DNA 
                 GTCTAGACTCGTGGT 
                 TTTTGGCCAGG 
                 [6FAM]CAATTTTCTAGG 
                 72° C. 10 s) × 45 cycles 
               
               
                   
                   
                   
                 GGA (SEQ ID NO: 25) 
                 ACAC (SEQ ID 
                 GGGAGCACCCAC[BHQ1] 
                   
               
               
                   
                   
                   
                   
                 NO: 26) 
                 (SEQ ID NO: 27) 
                   
               
               
                   
               
               
                 CpG Island 2 
                 1583-1622 
                   
                 HBV_F_1583_1602: 
                 HBV_R_1604_1622: 
                 SYBR Green 
                 95° C. 5 min, (95° C. 10 s 60° C. 
               
               
                   
                   
                   
                 ACTTCGCTTCACCTCT 
                 CACGGTGGT 
                   
                 10 s, 72° C. 10 s) × 45 cycles, melt [95° C. 
               
               
                   
                   
                   
                 GCAC (SEQ ID NO: 28) 
                 CTCCATGCTAC 
                   
                 5 s, 65° C. 60 s, 97° C. continuous], 
               
               
                   
                   
                   
                   
                 (SEQ ID NO: 29) 
                   
                 40° C. 30 s 
               
               
                   
                 1613-1671 
                   
                 HBV_F_1613_1629: 
                 HBVRev1671_1654_Chr21: 
                 SYBR Green 
                 95° C. 5 min, (95° C. 
               
               
                   
                   
                   
                 GACCACCGTGAACGC 
                 AGTCCAAGAGT 
                   
                 10 s 60° C. 10 s, 72° C. 10 s) × 
               
               
                   
                   
                   
                 CC (SEQ ID NO: 30) 
                 CCTGTTGTGCA 
                   
                 45 cycles, melt [95° C. 5 s, 65° C. 60 s, 
               
               
                   
                   
                   
                   
                 AGACCTT (SEQ 
                   
                 97° C. continuous], 40° C. 30 s 
               
               
                   
                   
                   
                   
                 ID NO: 31) 
                   
                   
               
               
                   
                 1633-1680 
                   
                 HBV_F_1633_1653: 
                 HBV_R_1660_1680: 
                 SYBR Green 
                 95° C. 5 min, (95° C. 
               
               
                   
                   
                   
                 AGGTCTTGCCCAAGG 
                 TTGCTGAGA 
                   
                 10 s 60° C. 10 s, 72° C. 10 s) × 
               
               
                   
                   
                   
                 TCTTAC (SEQ ID 
                 GTCCAAGAGTC 
                   
                 45 cycles, melt [95° C. 5 s, 65° C. 
               
               
                   
                   
                   
                 NO: 32) 
                 C (SEQ ID 
                   
                 60 s, 97° C. continuous], 40° C. 30 s 
               
               
                   
                   
                   
                   
                 NO: 33) 
                   
                   
               
               
                   
                 1685-1719 
                   
                 HBV_F_1685_1719: 
                 HBV_R_1713_1740: 
                 SYBR Green 
                 95° C. 5 min, (95° C. 
               
               
                   
                   
                   
                 AACGACCGACCTTGA 
                 CTCCTCCCAGT 
                   
                 10 s 60° C. 10 s, 72° C. 10 s) × 
               
               
                   
                   
                   
                 GGCATACTTC (SEQ ID 
                 CTTTAAACAAA 
                   
                 45 cycles, melt [95° C. 5 s, 65° C. 60 s, 
               
               
                   
                   
                   
                 NO: 34) 
                 CAGTC (SED ID 
                   
                 97° C. continuous], 40° C. 30 s 
               
               
                   
                   
                   
                   
                 NO: 35) 
                   
                   
               
               
                   
                 1741-1791 
                   
                 TRGGGGAGGAGATAA 
                 ATGCCTACAGC 
                 SYBR Green 
                 95° C. 5 min, (95° C. 10 s 65° C. 10 s, 
               
               
                   
                   
                   
                 GGTTAAAGGTC (SEQ 
                 CTCCTAGTACA 
                   
                 72° C. 8 s) × 40 cycles, melt 
               
               
                   
                   
                   
                 ID NO: 36) 
                 A (SEQ ID 
                   
                 [95° C. 5 s, 65° C. 60 s, 97° C. 
               
               
                   
                   
                   
                   
                 NO: 37) 
                 continuous], 40° C. 30 s 
                   
               
               
                   
               
               
                 CpG Island 3 
                 2294-2347 
                   
                 HBV gp4 Fwd3a1: 
                 HBV gp4 Rev3a: 
                 HBV gp4 Probe_53bp: 
                 95° C. 5 min, (95° C. 10 s 58° C. 15 s, 
               
               
                   
                   
                   
                 TACAGACCACCAAAT 
                 AACAACAGTA 
                 [6FAM]C+CTAT+C+T+T 
                 72° C. 15 s) × 45 cycles 
               
               
                   
                   
                   
                 GCC (SEQ ID NO: 38) 
                 GTTTCCGGAAG 
                 AT+CAA+CA+CTT+C[BHQ1] 
                   
               
               
                   
                   
                   
                   
                 TGTTG (SEQ ID 
                 (SEQ ID NO: 40) 
                   
               
               
                   
                   
                   
                   
                 NO: 39) 
               
               
                   
               
               
                 “+” indicates LNA nucleotide 
               
               
                 “R” indicates A + G nucleotides 
               
            
           
         
       
     
     Below is the detailed protocol for quantification of bisulfite converted CpG island 1 of the HBV genome (results shows in  FIG. 8 ). 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                   
                   
                 Product 
               
               
                 Primer/Probe 
                 Sequence: 5′ to 3′ 
                 Size 1   
               
               
                   
               
               
                 HBV_BSC1F1 (SEQ ID NO: 1) 
                 GGTTTTTTTTGTTGATAAGAATTT 
                 79 bp 
               
               
                   
               
               
                 HBV_BSC1R3 (SEQ ID NO: 2) 
                 CCCCCTAAAAAATTAAAAAAAA 
                   
               
               
                   
               
               
                 BSC1TQ (SEQ ID NO: 3) 
                 [6FAM]TTATAATATTATAGAGTTTAGATTYGTGGTGGA[BHQ1] 
               
               
                   
               
            
           
           
               
            
               
                 Roche Light Cycler 480 - Template: Mono-color Hydrolysis Probe 
               
               
                 95° C. 5 min, (95° C. 10 s 50° C. 15 s, 72° C. 10 s) × 50 cycles 
               
            
           
           
               
               
               
               
               
               
            
               
                 Component 
                 [Stock] 
                 [Final] 
                 uL/rxn 
                 DNA Template 
                 HBV Copy/ul 
               
               
                   
               
               
                 LC480 Probe Master Mix 
                 2x 
                 1x 
                 5 
                 HepG2 BSF (500 copies) 
                 0 
               
               
                 HBV_BSC1_F1R3 
                 10 uM 
                   1 uM 
                 1 
                 Meth. HepG2.215 
                 10 
               
               
                 Probe (BSC1TQ) 
                  1 uM 
                 0.1 uM 
                 1 
                 Meth. HepG2.215 
                 50 
               
               
                 H 2 O 
                 — 
                 — 
                 2 
                 Meth. HepG2.215 
                 100 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 Meth. HepG2.215 
                 1000 
               
               
                   
                   
                 Total: 
                 10 
                 Meth. HepG2.215 
                 10000 
               
               
                   
               
            
           
         
       
     
     Below is the detailed protocol for the quantification of methylated CpG island 1 of the HBV genome (results shows in  FIG. 9 ). 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 Primer/Probe 
                 Sequence: 5′ to 3′ 
                 Product 
               
               
                   
               
               
                 HBV_C1F2B (SEQ ID NO: 4) 
                 ACGTGTTTTGGTTAAAATTCGTAGTTTTTA 
                 110 bp 
               
               
                   
               
               
                 HBV_C1R5B (SEQ ID NO: 5) 
                 AATATAATAAAACGCCGCAAACACATC 
                   
               
               
                   
               
               
                 C1MSTQ (SEQ ID NO: 6) 
                 [6FAM]TTTTTTAATTTGTTTTGGTTATCGTTGGATG[BHQ1] 
               
               
                   
               
            
           
           
               
            
               
                 Roche Light Cycler 480 - Template: Mono-color Hydrolysis Probe 
               
               
                 95° C. 5 min, (95° C. 10 s 55° C. 30 s, 72° C. 10 s) × 50 cycles 
               
            
           
           
               
               
               
               
               
               
            
               
                 Component 
                 [Stock] 
                 [Final] 
                 uL/rxn 
                 DNA Template 
                 HBV copy/ul 
               
               
                   
               
               
                 LC480 Probe Master Mix 
                 2x 
                 1x 
                 5 
                 HepG2 BSF (500 copies) 
                 0 
               
               
                 HBV_C1F2B_R5B 
                 10 uM 
                   1 uM 
                 1 
                 Meth. HepG2.215 
                 1 
               
               
                 Probe (C1MSTQ) 
                  1 uM 
                 0.1 uM 
                 1 
                 Meth. HepG2.215 
                 5 
               
               
                 H 2 O 
                 — 
                 — 
                 2 
                 Meth. HepG2.215 
                 10 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 Meth. HepG2.215 
                 50 
               
               
                   
                   
                 Total: 
                 10 
                 Meth. HepG2.215 
                 100 
               
               
                   
                   
                   
                   
                 Meth. HepG2.215 
                 1000 
               
               
                   
               
            
           
         
       
     
     Below is the detailed protocol for the quantification of bisulfite converted CpG island 2 of the HBV genome (results shows in  FIG. 10 ). 
                                        Primer/Probe   Sequence: 5′ to 3′   Product               HBV_BSC2F1 (SEQ ID NO: 7)   GGATTTTTGGATTTTTAGTAATGTT   74 bp               HBV_BSC2R3 (SEQ ID NO: 8)   CCAATCTTTAAACAAACAATCTTTAA                   BSC2TQ (SEQ ID NO: 9)   [6FAM]ATGTTAAYGATYGATTTTGAGGTATATTTTAA[BHQ1]                         Roche Light Cycler 480 - Template: Mono-color Hydrolysis Probe       95° C. 5 min, (95° C. 10 s 53° C. 15 s, 72° C. 10 s) × 50 cycles                                     Component   [Stock]   [Final]   uL/rxn   DNA Template   HBV Copy/ul               LC480 Probe Master Mix   2x   1x   5   HepG2 BSF (500 copies)   0       HBV_BSC2_F1R3   10 uM     1 uM   1   Meth. HepG2.215   10       Probe (BSC2TQ)    1 uM   0.1 uM   1   Meth. HepG2.215   100       H 2 O   —   —   2   Meth. HepG2.215   500       DNA Template   —   —   1   Meth. HepG2.215   1000               Total:   10                    
Below is the detailed protocol for the quantification of methylated CpG island 2 of the HBV genome (results shows in  FIG. 11 ).
 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                   
                 Primer 
                 Sequence: 5′ to 3′ 
                 Product 
               
               
                   
               
               
                   
                 HBV_C2MF3 (SEQ ID: 10) 
                 TGTCGTTTCGGTCGATTAC 
                 78 bp 
               
               
                   
               
               
                   
                 HBV_C2MR3 (SEQ ID NO: 11) 
                 CACGATCCGACAAATAAAAA 
               
               
                   
               
            
           
           
               
            
               
                 Roche Light Cycler 480 - Template: SYBR Green I 
               
               
                 95° C. 5 min, (95° C. 10 s 52° C. 30 s, 72° C. 10 s) × 45 cycles, Melting curve, 40° C. 30 S 
               
            
           
           
               
               
               
               
               
               
            
               
                 Component 
                 [Stock] 
                 [Final] 
                 uL/reaction 
                 DNA Template 
                 HBV copy/ul 
               
               
                   
               
               
                 LC480 SYBR Green 
                 2x 
                 1x 
                 5 
                 HepG2 BSF (500 copies) 
                 0 
               
               
                 Mix 
                   
                   
                   
                 Meth. HepG2.215 
                 5 
               
               
                 HBV_C2MF3_R3 
                 10 uM 
                 1 uM 
                 1 
                 Meth. HepG2.215 
                 10 
               
               
                 H 2 O 
                 — 
                 — 
                 3 
                 Meth. HepG2.215 
                 100 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 Meth. HepG2.215 
                 1000 
               
               
                   
                   
                 Total: 
                 10 
                 Meth. HepG2.215 
                 10000 
               
               
                   
                   
                   
                   
                 Meth. HepG2.215 
                 20000 
               
               
                   
               
            
           
         
       
     
     Below is the detailed protocol for the quantification of bisulfate converted CpG island 3 of the HBV genome (results shows in  FIG. 12 ). 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 Primer Name 
                 Sequence: 5′ to 3′ 
                 Product 
               
               
                   
               
               
                 HBV_BSC3F9 (SEQ ID NO: 12) 
                 TTATGTTAATGTTAATATGGGTTTAAA 
                 79 bp 
               
               
                   
               
               
                 HBV_BSC3R9.10 (SEQ ID NO: 13) 
                 TTCTCTTCCAAAAATAAAACAA 
                   
               
               
                   
               
               
                 C3BSTQ (SEQ ID NO: 14) 
                 6FAM-TTAgATAATTATTg+Tgg+T+T+T+TA+TA+T--BBQ 
               
               
                   
               
            
           
           
               
            
               
                 Roche Light Cycler 480 - Template: Mono Color Hydrolysis Probe I 
               
               
                 95° C. 10 min, (95° C. 10 s, 52° C. 30 s, 72° C. 10 s) × 50 cycles 
               
            
           
           
               
               
               
               
               
               
            
               
                 Component 
                 [Stock] 
                 [Final] 
                 uL/rxn 
                 DNA Template 
                 HBV copy/ul 1   
               
               
                   
               
               
                 LC480 Probe Master Mix 
                 2x 
                 1x 
                 5 
                 water 
                 0 
               
               
                 HBV_BF9_R9.10 
                 10 uM 
                   1 uM 
                 1 
                 HepG2 BSF (500 copies) 
                 0 
               
               
                 Probe (C3BSTQ) 
                  1 uM 
                 0.1 uM 
                 1 
                 Meth. HepG2.215 
                 50 
               
               
                 H 2 O 
                 — 
                 — 
                 2 
                 Meth. HepG2.215 
                 100 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 Meth. HepG2.215 
                 1000 
               
               
                   
                   
                 Total: 
                 10 
                 Meth. HepG2.215 
                 10000 
               
               
                   
               
            
           
         
       
     
     Below is the detailed protocol for the quantification of methylated CpG island 3 of the HBV genome (results shows in  FIG. 13 ). 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                   
                 Primer Name 
                 Sequence: 5′ to 3′ 
                 Product 
               
               
                   
               
               
                   
                 HBV_MSP2F (SEQ ID NO: 15) 
                 GTGTGGATTCGTATTTTTTTC 
                 143 bp 
               
               
                   
               
               
                   
                 HBV_MSP2R (SEQ ID NO: 16) 
                 GACGATTAAAACCTTCGTCT 
                   
               
               
                   
               
               
                   
                 C3AMSTQ (SEQ ID NO: 17) 
                 AACCTACCTCGTCGTCTAACAACAAT 
               
               
                   
               
            
           
           
               
            
               
                 Roche Light Cycler 480 - Template: Mono Color Hydrolysis Probe I 
               
               
                 95° C. 10 min, (95° C. 10 s, 53° C. 30 s, 72° C. 10 s) × 45 cycles 
               
            
           
           
               
               
               
               
               
               
            
               
                 Component 
                 [Stock] 
                 [Final] 
                 uL/rxn 
                 DNA Template 
                 HBV Copy/ul 
               
               
                   
               
               
                 LC480 Probe Master Mix 
                 2x 
                 1x 
                 5 
                 water 
                 0 
               
               
                 HBV_MSP2F_2R 
                 10 uM 
                   1 uM 
                 1 
                 HepG2 BSF (500 copies) 
                 0 
               
               
                 Probe (C3AMSTQ) 
                  1 uM 
                 0.1 uM 
                 1 
                 Meth. HepG2.215 
                 50 
               
               
                 H 2 O 
                 — 
                 — 
                 2 
                 Meth. HepG2.215 
                 100 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 Meth. HepG2.215 
                 1000 
               
               
                   
                   
                 Total: 
                 10 
                 Meth. HepG2.215 
                 10000 
               
               
                   
               
            
           
         
       
     
     Below is the detailed protocol for detection of circulation derived methylated CpG island 3 of the HBV genome by using HBV-MSP Short Amplicon Assay (mHBV) Target Amplicon Size: 42 bp (results shows in  FIG. 14 ). 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 Primer Name 
                 Sequence: 5′ to 3′ 
                 Product 
               
               
                   
               
               
                 HBV_SMF1 (SEQ ID NO: 18) 
                 GCTCTTCGTGGTGTGGTGAAGAAGAATTTTTTCGTTTC 
                 42 bp 
               
               
                   
               
               
                 HBV_MSP2R (SEQ ID NO: 16) 
                 GACGATTAAAACCTTCGTCT 
                   
               
               
                   
               
               
                 HBV_S2F1 (SEQ ID NO: 19) 
                 TGTGGTGAAGAAGAATT 
                   
               
               
                   
               
               
                 HBV_S2R1 (SEQ ID NO: 20) 
                 GACGATTAAAACCTTC 
               
               
                   
               
            
           
           
               
            
               
                 1st PCR: Product size - 60 bp 
               
            
           
           
               
               
               
               
            
               
                 Component 
                 Stock Concentration 
                 Final Concentration 
                 uL/reaction 
               
               
                   
               
               
                 10X PCR Buffer 
                 10X 
                 1X 
                 1 
               
            
           
           
               
               
               
               
               
               
            
               
                 dNTP 
                 2.5 
                 mM each 
                 250 
                 uM each 
                 0.8 
               
               
                 HBT_SMF1/MSP2R 
                 10 
                 uM 
                 1 
                 uM 
                 1 
               
               
                 HotStart Taq Plus 
                 5 
                 U/uL 
                 1 
                 U/reaction 
                 0.2 
               
            
           
           
               
               
               
               
            
               
                 DNA Template 
                 — 
                 — 
                 1 
               
               
                 H 2 O 
                 — 
                 — 
                 6 
               
               
                 Total 
                   
                   
                 10 
               
               
                   
               
            
           
           
               
            
               
                 Eppendorf Thermal Cycler: 95° C. 5 min, (95° C. 30 s, 50° C. 30 s, 
               
               
                 72° C. 30 s) × 27 cycles, 72° C. 4 min, 4° C. Hold 
               
               
                 *Dilute 1st PCR products 1:10 by adding 90 uL H 2 O to each tube. 
               
               
                 2nd PCR: Product size - 45 bp 
               
            
           
           
               
               
               
               
            
               
                 Component 
                 Stock Concentration 
                 Final Concentration 
                 uL/reaction 
               
               
                   
               
               
                 LC480 SYBR Green Mix 
                 2X 
                 1X 
                 5 
               
               
                 HBV_S2F1/R1 
                 10 uM 
                 1 uM 
                 1 
               
               
                 1st PCR Product (1:10) 
                 — 
                 — 
                 1 
               
               
                 H 2 O 
                 — 
                 — 
                 3 
               
               
                 Total 
                   
                   
                 10 
               
               
                   
               
            
           
           
               
            
               
                 Roche LightCycler 480 - Template: SYBR Green I 
               
               
                 95° C. 5 min, (95° C. 10 s, 50° C. 15 s, 72° C. 10 s) × 45 cycles, 
               
               
                 Melting curve, 40° C. 30 s 
               
            
           
         
       
     
     Below is the detailed protocol for quantification of methylated CpG island 3 of the HBV genome by a short amplicon MSP assay that can be used for fragmented DNA (results shows in  FIG. 15 ). 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                   
                 Primer Name 
                 Sequence: 5′ to 3′ 
                 Product 
               
               
                   
               
               
                   
                 C3.M_F1 (SEQ ID NO: 21) 
                 AGAATTTTTTCGTTTCGTAGAC 
                 55 bp 
               
               
                   
               
               
                   
                 C3.M_R3 (SEQ ID NO: 22) 
                 AAAATCTTCTACGACGCGACGATTAA 
                   
               
               
                   
               
               
                   
                 C3.M_R2 (SEQ ID NO: 23) 
                 CTACGACGCGACGATTAAAAC 
                   
               
               
                   
               
               
                   
                 CG3.MSP SP (SEQ ID NO: 24) 
                 FLQ-TCgTTTCgTAgACgAAggT--PH 
               
               
                   
               
            
           
           
               
            
               
                 First Step: 
               
               
                 Hotstart TAQ Plus Amplification: Eppendorf Vapo Protect Thermocycler - Program 
               
               
                 Template: Hotstart 95° C. 5 min, (95° C. 30 s, 50° C. 30 s, 
               
               
                 72° C. 30 s) × 27 Pause + 40 cycles; 72° C. 4 min, 
               
               
                 4° C. hold 
               
               
                 After 27 cycles, take out 1 uL and dilute with 9 ul H2O (2nd step template), 
               
               
                 put back remaining reaction volume for 40 cycles (melt template) 
               
               
                 Second step: 
               
               
                 Roche Light Cycler 480 - Template: SybrGreen 
               
               
                 95° C. 5 min, (95° C. 10 s, 53° C. 30 s, 72° C. 10 s) × 45 cycles 
               
               
                 Melt step: 
               
               
                 Roche Light Cycler 480 - Template: Simple Probe 
               
               
                 95° C. 1 min, 95° C. 60 s, 40° C. 120 s, 95° C. continuous; 40° C. 30 s 
               
            
           
           
               
               
            
               
                 Hotstart TAQ Plus Amplification 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Component 
                 [Stock] 
                 [Final] 
                 uL/rxn 
                 DNA Template BSF 
                 HBV Copy/ul 
               
               
                   
               
               
                 HotStart TAQ Plus 
                 2x 
                 1x 
                 5 
                 water 
                 0 
               
               
                 C3.M_F1/R3 
                 10 uM 
                 1 uM 
                 1 
                 Meth. HepG2 (200 copies) 
                 0 
               
               
                 H 2 O 
                 — 
                 — 
                 1 
                 Meth. HepG2.215* 
                 3 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 Meth. HepG2.215* 
                 10 
               
               
                   
                   
                 Total: 
                 10 
                 Meth. HepG2.215* 
                 100 
               
               
                   
                   
                   
                   
                 Meth. HepG2.215* 
                 1000 
               
               
                   
                   
                   
                   
                 Meth. HepG2.215* 
                 10000 
               
               
                   
               
            
           
           
               
            
               
                 *In background of Meth. HepG2 200 cp 
               
            
           
           
               
               
            
               
                 Sybrgreen qPCR (2nd step) 
                 Simple Probe Melt 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Component 
                 [Stock] 
                 [Final] 
                 uL/rxn 
                 Component 
                 [Stock] 
                 [Final] 
                 uL/rxn 
               
               
                   
               
               
                 LC480 Sybrgreen 
                 2x 
                 1x 
                 5 
                 CG3.MSP SP 
                 2 uM 
                 0.2 uM 
                 1 
               
               
                 Master 
                   
                   
                   
                 H 2 O 
                 — 
                 — 
                 5 
               
               
                 C3.M_F1/R2 
                 10 uM 
                 1 uM 
                 1 
                 DNA Template 
                 — 
                 — 
                 4 
               
               
                 H 2 O 
                 — 
                 — 
                 1 
                 (Hotstart TAQ 
                   
                   
                   
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 amplification 
                   
                   
                   
               
               
                   
                   
                 Total: 
                 10 
                 PCR product) 
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Total: 
                 10 
               
               
                   
               
            
           
         
       
     
     Below is the detailed protocol for quantification of genomic CpG island 1 of the HBV genome (results shows in  FIG. 16 ). 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                   
                   
                   
                 Product 
               
               
                   
                 Primer/Probe 
                 Sequence: 5′ to 3′ 
                 Size 
               
               
                   
               
               
                   
                 HBV240F3 (SEQ ID NO: 25) 
                 GTCTAGACTCGTGGTGGA 
                 64 bp 
               
               
                   
               
               
                   
                 HBVC240R1 (SEQ ID NO: 26) 
                 TTTTGGCCAGGACAC 
                   
               
               
                   
               
               
                   
                 HBV240 TaqMan (SEQ ID NO: 27) 
                 [6FAM]CAATTTTCTAGGGGGAGCACCCAC[BHQ1] 
               
               
                   
               
            
           
           
               
            
               
                 Roche Light Cycler 480 - Template: Mono-color Hydrolysis Probe 
               
               
                 95° C. 5 min, (95° C. 10 s 54° C. 10 s, 64° C. 15 s) × 45 cycles 
               
            
           
           
               
               
               
               
               
               
            
               
                 Component 
                 [Stock] 
                 [Final] 
                 uL/rxn 
                 DNA Template 
                 HBV Copy/ul 
               
               
                   
               
               
                 LC480 Probe Master Mix 
                 2x 
                 1x 
                 5 
                 PSP65 HBV + HepG2 0.1 ng/ul 
                 1.5E8 
               
               
                 HBV240F3/C240R1 
                 10 uM 
                   .5 uM 
                 0.5 
                 PSP65 HBV + HepG2 0.1 ng/ul 
                 1.5E7 
               
               
                 Probe (HBV240 TaqMan) 
                  3 uM 
                 0.15 uM 
                 0.5 
                 PSP65 HBV + HepG2 0.1 ng/ul 
                 1.5E6 
               
               
                 H 2 O 
                 — 
                 — 
                 2 
                 PSP65 HBV + HepG2 0.1 ng/ul 
                 1.5E5 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 PSP65 HBV + HepG2 0.1 ng/ul 
                 1.5E4 
               
               
                   
                   
                 Total: 
                 10 
                 PSP65 HBV + HepG2 0.1 ng/ul 
                 1.5E3 
               
               
                   
                   
                   
                   
                 PSP65 HBV + HepG2 0.1 ng/ul 
                 1.5E2 
               
               
                   
                   
                   
                   
                 PSP65 HBV + HepG2 0.1 ng/ul 
                 1.5E1 
               
               
                   
                   
                   
                   
                 PSP65 HBV + HepG2 0.1 ng/ul 
                 1.5 
               
               
                   
                   
                   
                   
                 H2O + HepG2 
               
               
                   
               
            
           
         
       
     
     Below is the detailed protocol for quantification of genomic CpG island 2 of the HBV genome (results shows in  FIG. 17 ). 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                   
                 Primer/Probe 
                 Sequence: 5′ to 3′ 
                 Product Size 
               
               
                   
               
               
                   
                 HBV_F_1583_1602 (SEQ ID NO: 28) 
                 ACTTCGCTTCACCTCTGCAC 
                 39 bp 
               
               
                   
               
               
                   
                 HBV_R_1604_1622 (SEQ ID NO: 29) 
                 CACGGTGGTCTCCATGCTAC 
               
               
                   
               
            
           
           
               
            
               
                 Roche Light Cycler 480 - Template: SYBR Green 
               
               
                 95° C. 5 min, (95° C. 10 s 60° C. 10 s, 72° C. 10 s) × 45 cycles, melt [95° C. 5 s, 65° C. 60 s, 97° C. continuous, 40 C. 30 s 
               
            
           
           
               
               
               
               
               
               
            
               
                 Component 
                 [Stock] 
                 [Final] 
                 uL/rxn 
                 DNA Template 
                 HBV Copy/ul 
               
               
                   
               
               
                 LC480 SYBR Green 
                 2x 
                 1x 
                 5 
                 pspHBV 1000 cp + HepG2 1 ng 
                 1000 
               
               
                 HBV 
                 10 uM 
                 1 uM 
                 1 
                 pspHBV 100 cp + HepG2 1 ng 
                 100 
               
               
                 F1583_1603/R_1604_1622 
                   
                   
                   
                 pspHBV 10 cp + HepG2 1 ng 
                 10 
               
               
                 H 2 O 
                 — 
                 — 
                 3 
                 pspHBV 1 cp + HepG2 1 ng 
                 1 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 H2O 
                   
               
               
                   
                   
                 Total: 
                 10 
               
               
                   
               
            
           
         
       
     
     Below is the detailed protocol for quantification of genomic CpG island 2 of the HBV genome (results shows in  FIG. 18 ). 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                   
                   
                 Product 
               
               
                 Primer/Probe 
                 Sequence: 5′ to 3′ 
                 Size 
               
               
                   
               
               
                 HBV_F_1613_1629 (SEQ ID NO: 30) 
                 GACCACCGTGAACGCCC 
                 58 bp 
               
               
                   
               
               
                 HBVRev1671_1654_Chr21 (SEQ ID NO: 31) 
                 AGTCCAAGAGTCCTGTTGTGCAAGACCTT 
               
               
                   
               
            
           
           
               
            
               
                 Roche Light Cycler 480 - Template: SYBR Green 
               
               
                 95° C. 5 min, (95° C. 10 s 60° C. 10 s, 72° C. 10 s) × 45 cycles, melt [95° C. 5 s, 65° C. 60 s, 97° C. continuous], 40 C. 30 s 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 uL/ 
                   
                   
               
               
                 Component 
                 [Stock] 
                 [Final] 
                 rxn 
                 DNA Template 
                 HBV Copy/ul 
               
               
                   
               
               
                 LC480 SYBR Green 
                 2x 
                 1x 
                 5 
                 pspHBV 1000 cp + HepG2 1 ng 
                 1000 
               
               
                 HBV_F_1613_1629/ 
                 10 uM 
                 1 uM 
                 1 
                 pspHBV 100 cp + HepG2 1 ng 
                 100 
               
               
                 HBVRev1671_1654_Chr21 
                   
                   
                   
                 pspHBV 10 cp + HepG2 1 ng 
                 10 
               
               
                 H 2 O 
                 — 
                 — 
                 3 
                 pspHBV 1 cp + HepG2 1 ng 
                 1 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 H2O 
                   
               
               
                   
                   
                 Total: 
                 10 
               
               
                   
               
            
           
         
       
     
     Below is the detailed protocol for quantification of genomic CpG island 2 of the HBV genome (results shows in  FIG. 19 ). 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                   
                 Primers 
                 Sequence: 5′ to 3′ 
                 Product Size 
               
               
                   
               
               
                   
                 HBV_F_1633_1653 (SEQ ID NO: 32) 
                 AGGTCTTGCCCAAGGTCTTAC 
                 47 bp 
               
               
                   
               
               
                   
                 HBV_R_1660_1680 (SEQ ID NO: 33) 
                 TTGCTGAGAGTCCAAGAGTCC 
               
               
                   
               
            
           
           
               
            
               
                 Roche Light Cycler 480 - Template: SYBR Green 
               
               
                 95° C. 5 min, (95° C. 10 s 60° C. 10 s, 72° C. 10 s) × 45 cycles, 
               
               
                 melt [95° C. 5 s, 65° C. 60 s, 97° C. continuous], 40 C. 30 s 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 uL/ 
                   
                   
               
               
                 Component 
                 [Stock] 
                 [Final] 
                 rxn 
                 DNA Template 
                 HBV Copy/ul 
               
               
                   
               
               
                 LC480 SYBR Green 
                 2x 
                 1x 
                 5 
                 pspHBV 1000 cp + HepG2 
                 1000 
               
               
                 HBV 
                 10 uM 
                 1 uM 
                 1 
                 1 ng 
                   
               
               
                 F1633_1653/R_1660_1680 
                   
                   
                   
                 pspHBV 100 cp + HepG2 1 ng 
                 100 
               
               
                 H 2 O 
                 — 
                 — 
                 3 
                 pspHBV 10 cp + HepG2 1 ng 
                 10 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 pspHBV 1 cp + HepG2 1 ng 
                 1 
               
               
                   
                   
                 Total: 
                 10 
                 H2O 
               
               
                   
               
            
           
         
       
     
     Below is the detailed protocol for quantification of genomic CpG island 2 of the HBV genome (results shows in  FIG. 20 ). 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                   
                   
                   
                 Product 
               
               
                   
                 Primer/Probe 
                 Sequence: 5′ to 3′ 
                 Size 
               
               
                   
               
               
                   
                 HBV_F_1685_1719 (SEQ ID NO: 34) 
                 AACGACCGACCTTGAGGCATACTTC 
                 55 bp 
               
               
                   
               
               
                   
                 HBV_R_1713_1740 (SEQ ID NO: 35) 
                 CTCCTCCCAGTCTTTAAACAAACAGTC 
               
               
                   
               
            
           
           
               
            
               
                 Roche Light Cycler 480 - Template: SYBR Green 
               
               
                 95° C. 5 min, (95° C. 10 s 60° C. 10 s, 72° C. 10 s) × 45 cycles, 
               
               
                 melt [95° C. 5 s, 65° C. 60 s, 97° C. continuous], 40 C. 30 s 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 uL/ 
                   
                 HBV 
               
               
                 Component 
                 [Stock] 
                 [Final] 
                 rxn 
                 DNA Template 
                 Copy/ul 
               
               
                   
               
               
                 LC480 SYBR Green 
                 2x 
                 1x 
                 5 
                 pspHBV 100 cp + HepG2 1 ng 
                 100 
               
               
                 HBV_F_1685_1719/R_1713_1740 
                 10 uM 
                 1 uM 
                 1 
                 pspHBV 10 cp + HepG2 1 ng 
                 10 
               
               
                 H 2 O 
                 — 
                 — 
                 3 
                 pspHBV 1 cp + HepG2 1 ng 
                 1 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 H2O 
                   
               
               
                   
                   
                 Total: 
                 10 
               
               
                   
               
            
           
         
       
     
     Below is the detailed protocol for quantification of genomic CpG island 2 of the HBV genome (results shows in  FIG. 21 ). 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                   
                 Primer/Probe 
                 Sequence: 5′ to 3′ 
                 Product Size 
               
               
                   
               
               
                   
                 HBV_F_1741_1767 (SEQ ID NO: 36) 
                 TRGGGGAGGAGATAAGGTTAAAGGTC 
                 50 bp 
               
               
                   
               
               
                   
                 HBV_R_1768_1791 SEQ ID NO: 37) 
                 ATGCCTACAGCCTCCTAGTACAA 
               
               
                   
               
            
           
           
               
            
               
                 Roche Light Cycler 480 - Template: SYBR Green 
               
               
                 95° C. 5 min, (95° C. 10 s 65° C. 10 s, 72° C. 8 s) × 40 cycles, 
               
               
                 melt [95° C. 5 s, 65° C. 60 s, 97° C. continuous], 40 C. 30 s 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 uL/ 
                   
                   
               
               
                 Component 
                 [Stock] 
                 [Final] 
                 rxn 
                 DNA Template 
                 HBV Copy/ul 
               
               
                   
               
               
                 LC480 Sybrgreen Master 
                 2x 
                 1x 
                 5 
                 PSP65 HBV + HepG2 1 ng/ul 
                 1.0E4 
               
               
                 HBV_F_1741_1767/R_1768_1791 
                 10 uM 
                 1 uM 
                 1 
                 PSP65 HBV + HepG2 1 ng/ul 
                 1.0E3 
               
               
                 H 2 O 
                 — 
                 — 
                 3 
                 PSP65 HBV + HepG2 1 ng/ul 
                 1.0E2 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 PSP65 HBV + HepG2 1 ng/ul 
                 1.0E1 
               
               
                   
                   
                 Total: 
                 10 
                 H2O 
               
               
                   
               
            
           
         
       
     
     Below is the detailed protocol for quantification of genomic CpG island 3 of the HBV genome (results shows in  FIG. 22 ). 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                   
                   
                 Product 
               
               
                 Primer/Probe 
                 Sequence: 5′ to 3′ 
                 Size 
               
               
                   
               
               
                 HBV gp4 Fwd3a1 (SEQ ID NO: 38) 
                 TACAGACCACCAAATGCC 
                 54 bp 
               
               
                   
               
               
                 HBV gp4 Rev3a (SEQ ID NO: 39) 
                 AACAACAGTAGTTTCCGGAAGTGTTGA 
                   
               
               
                   
               
               
                 HBV gp4 Probe_53bp (SEQ ID NO: 40) 
                 [6FAM] C+CTAT+C+T+TA T+CAA+CA+CTT+C 
                   
               
               
                   
                 [BHQ1] 
               
               
                   
               
            
           
           
               
            
               
                 “+” indicates LNA nucleotide 
               
               
                 Roche Light Cycler 480 - Template: Mono-color Hydrolysis Probe 
               
               
                 95° C. 5 min, (95° C. 10 s 65° C. 10 s, 72° C. 15 s) × 40 cycles 
               
            
           
           
               
               
               
               
               
               
            
               
                 Component 
                 [Stock] 
                 [Final] 
                 uL/rxn 
                 DNA Template 
                 HBV Copy/ul 
               
               
                   
               
               
                 LC480 Probe Master 
                 2x 
                 1x 
                 5 
                 PSP65 HBV + HepG2 1 ng/ul 
                 1.0E7 
               
               
                 Mix 
                   
                   
                   
                 PSP65 HBV + HepG2 1 ng/ul 
                 1.0E6 
               
               
                 HBV240F3/C240R1 
                 10 uM 
                    1 uM 
                 1 
                 PSP65 HBV + HepG2 1 ng/ul 
                 1.0E5 
               
               
                 Probe (HBV240 
                  2 uM 
                 0.15 uM 
                 .75 
                 PSP65 HBV + HepG2 1 ng/ul 
                 1.0E4 
               
               
                 TaqMan) 
                   
                   
                   
                 PSP65 HBV + HepG2 1 ng/ul 
                 1.0E3 
               
               
                 H 2 O 
                 — 
                 — 
                 2.25 
                 PSP65 HBV + HepG2 1 ng/ul 
                 1.0E2 
               
               
                 DNA Template 
                 — 
                 — 
                 1 
                 PSP65 HBV + HepG2 1 ng/ul 
                 1.0E1 
               
               
                   
                   
                 Total: 
                 10 
                 PSP65 HBV + HepG2 1 ng/ul 
                 1.0 
               
               
                   
                   
                   
                   
                 HepG2 1 ng/ul 
                   
               
               
                   
                   
                   
                   
                 H2O 
               
               
                   
               
            
           
         
       
     
     All references cited in this application are incorporated by reference into this application in their entireties. 
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