Detection Of HBV CccDNA P Region And Its Clinical Significance

The first partESTABLISHMENT OF AMPLIFICATION AND EXTRACTION FOR FULL-LENGTH HEPATITIS B VIRUS COVALENTLY CLOSED CIRCULAR DNAObjective:To establish an assay for amplifying and extracting full-le ngth HBV cccDNA.Methods:(1)The QIAamp DNA Mini Kit were used to extract HBV tDNA.The extracted products were further purified with plasmid-safe ATP-dependent DNase;(2) Full-length HBV cccDNA of chronic hepatitis B p atients was amplified with RCA and specificy primers of HBV cccDNA (3) The products of RCA were amplified with a real-time PCR method an d specificy primers cross HBV gap region.Results:(1)Standard plasmid containing103-1010copies/ml,53samples from liver tissue of chronic hepatitis B which including4liver tissue w with serum HBV DNA<1.0×103copies/ml were amplified with RCA and specificy primers of HBV cccDNA, the detecble rate of3.2kb HBV c ccDNA were100%;(2)10liver tissue of non-chronic hepatitis B and10s erum of chronic hepatitis B were amplified with RCA and specificy prime rs of HBV cccDNA, the detecble rate of3.2kb HBV cccDNA were0%;(3) the products of RCA from53CHB liver tissue were detected with a real-time PCR method and specificy primers crossing HBV gap regio n, the detecable rate of cccDNA were100%.Conclution:hepatocyte full-length HBV cccDNA was amplified with HBV cccDNA specificy primers rolling circle amplification with high spec ificity and sencitivity The second partCLINICAL SIGNIFICANCE OF HEPATOCYTE HBV cccDNA POLYMERASE MUTATION DETECTION FOR NAs RESISTANT PATIENTSObjective:To probe the clinical significance of detected Hepatocyte HBV cccDNA Polymerase mutation for NAs resistant patients..Methods:Patients of3groups were all chronic hepatits B patients,N As-resistant group1were29patients who have occurred virological break through;NAs-resistant group2were4, LAM-naive resistant, then NAs-retr eatment reached therapy endpoint of China CHB guideline, and then recur rence; control group was20patients who have never therapied by antivira1.Test methods:HBV genotype were detected by nest PCR with multi prime rs;serum rcDNA were extracted by water boiling, hepatocyte full-length H BV cccDNA were amplified with HBV cccDNA special primers RCA; P gene mutation sites were detected with HBV Polymerase priers multi nest PCR and products sequence.Results:(1) NAs-resistant group1,the detectcable rate of M204I/V、L180M、A181T in hepatocyte HBV cccDNA were31%、20.7%、3.4%respec tively,in serum rcDNA were31%、27.6%、3.4%respectively;(2) NAs-resist ant group l,the detectable rate of S202G、V173L、T184I、V207L、A222T in hepatocyte HBV cccDNA were6.9%、3.4%、6.9%、10.3%、6.9%res pectively,in serum rcDNA were3.4%、3.4%、6.9%、6.9%、10.3%respetiv ely;(3)8mutation sites were not reported:N134E、Y151F、P177R、R192L、R193L、V253T、T259S、A223S;(4)The same detectable rate of hepato cyte HBV cccDNA and serum rcDNA mutation in the same patient was44.83%;(5) NAs-resistant group2, the mutation sites first-resistant serum rc DNA were the same as recurrence serum rcDNA in2patients;the mutatio n sites were same in hepatocyte HBV cccDNA of therapy endpoint as fir st-resistant and recurrence serum rcDNA in2patients.Conclusion:(1) The polymerase gene mutation sites and frequency we re corresponded with clinical history used RCA amplified hepatocyte full-1ength HBV cccDNA and sequencing;(2)Detection of polymerase gene mu tation of hepatocyte HBV cccDNA can be as a diagnosis of genotype resi stant,especially when serum DNA<1.0×103copies/ml;(4) RCA amplified he patocyte full-length HBV cccDNA with DNA sequencing can be help to e xplore the mechanism of NAs-resistant and the transformation kinetics of genotype resistant.