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Establishment of a Novel Assay for Detecting Mutations Associated with Antiviral Resistance of HBV Covalently Closed Circular DNA
http://www.tumorres.com/tumor-immunology/18386.htm
19
Dec
Establishment of a Novel Assay for Detecting Mutations Associated with Antiviral Resistance of HBV Covalently Closed Circular DNA and Its Application
posted by: Tumor Research
[Abstract]
This research was composed of two parts:Ⅰ. Establishment of a novel assay for detecting mutations associated with antiviral resistance of HBV covalently closed circular DNA.Ⅱ. Detection and comparison of mutations associated with antiviral resistance of cccDNA in liver cells and HBV DNA in peripheral serum of patients chronically infected with hepatitis B virus.Ⅰ. Establishment of a novel assay for detecting mutations associated with antiviral resistance of HBV covalently closed circular DNAObjective To establish an assay for detecting mutations associated with antiviral resistance of hepatitis B virus covalently closed circular DNA(HBV cccDNA). Methods①A selective Polymerase chain reaction (PCR) system based on primers set spanning the two gaps(DR regions) in HBV genome was established to preferentially amplify the region encoding lamivudine, adefovir and enticavir resistant substitutions of HBV cccDNA. To improve the sensivity of PCR, the PCR conditions such as the annealing temperature, the concentration of Mg2+ and Dimethyl sulfoxide(DMSO) were optimized. And the efficiency of this system to discriminate cccDNA and rcDNA was verified with HBV DNA molecules in peripheral serum and purified plasmid containing whole HBV DNA sequence (cccDNA).②The co-precipitation of Sodium Dodecyl Sulfate (SDS) and protein combining with the QIAamp DNA Mini Kit(tissue) were used to selectively extract HBV cccDNA. The extracted products were further purified with plasmid-safe ATP-dependent DNase(PSAD). HBV DNA and cccDNA in both supernatant and sediment were quantified using Real-time quantitative Polymerase Chain Reaction (Real Time PCR), and the impact of proteinase K on extracting efficiency was evaluated. ③The impact of DMSO on PCR was tested by comparison of the DNA sequencing results of the routine PCR with standard buffer and the nested PCR with buffer containing DMSO. Results①In this novel PCR system, the optimized concentration of Mg2+ and DMSO were 2mM and 2%. No detectable fluorescent signal was observed when the HBV DNA in peripheral serum samples were about 5x 106 copies per millilitre. The detectable HBV cccDNA was as low as 103 copies per millilitre at least by this method.②HBV DNA in peripheral serum was declined about 21og10 post hydrolyzed, and no impact of PS AD on cccDNA was detected.③When using co-precipitation of SDS and protein combining with the QIAamp DNA Mini Kit, the absence of proteinase K in digestive juice improved the yield of HBV rcDNA in the sedimentation, therefore raised the differentiation between rcDNA and cccDNA. And it didn’t impact the yield of HBV cccDNA in the superstratum.④The DNA sequencing results of nested PCR with buffer containing DMSO was consistent with the routine PCR with standard buffer. Conclusions Selective extraction of HBV cccDNA based on the co-precipitation of SDS-protein and the QIAamp DNA Mini Kit, and further purification with PSAD, integrating with selective PCR system can ensure the specific amplification of HBV cccDNA. The nested PCR with buffer containing 2% DMSO has no impact on DNA sequencing results. This method can be utilized in detection of mutations associated with antiviral resistance of intracellular HBV cccDNA. It is convenient, highly specific and highly sensitive.Ⅱ. Detection and comparison of mutations associated with antiviral resistance of cccDNA in liver cells and HBV DNA in peripheral serum of patients chronically infected with hepatitis B virus.Objective To determine the existence of mutations associated with antiviral resistance of cccDNA in liver cells of chronic HBV infected patients, and study the differences of virus strains in the liver cells and peripheral. Methods①Forty patients chronically infected with hepatitis B virus were investigated. Serum before operation and non-tumor liver tissues were selected.②The serum HBV DNA was extracted by directly boiling.③The HBV cccDNA and rcDNA were selectively extracted by co-precipitation using SDS-protein and the QIAamp DNA Mini Kit, and further purification with PSAD. To assess the effect of extraction and purification, the whole products were detected by real-time fluorescent quantitative PCR.④All the samples were amplified by PCR or nested PCR with the corresponding primers, and then the positive PCR products were sequenced commercialy. Results①Among the whole 40 patients, HBV DNA were detected in serum in 21 patients, HBV cccDNA were positive in liver cells in 31 patients, and HBV rcDNA were positive in liver cells in 37 patients. DNA sequences of these samples were successfully tested.②Mutations of rtM204I were detected in intracellular HBV cccDNA, rcDNA and serum HBV DNA of 2 patients. Mutations of rtM204I, rtQ215H were detected in intracellular HBV cccDNA and rcDNA of 2 patient, while the corresponding mutation was not detected in serum HBV DNA. Mutations of rtM204V, rtM204V, rtV173L+rtL180M+rtM204V were detected respectively in serum HBV DNA in 3 patients, while the corresponding mutants were not detected in intracellular HBV cccDNA or rcDNA. There also existed some other mutants on the various HBV cccDNA base sites. Conclusions Our results suggested that there are mutations associated with antiviral resistance of intracellular hepatitis B virus cccDNA in patients chronically infected with HBV. And the dominant virus quasispecies are different between HBV in liver cells and peripheral serum. There are more than one templates of HBV in liver cells.
Title: Establishment of a Novel Assay for Detecting Mutations Associated with Antiviral Resistance of HBV Covalently Closed Circular DNA and Its Application
19
Dec
Establishment of a Novel Assay for Detecting Mutations Associated with Antiviral Resistance of HBV Covalently Closed Circular DNA and Its Application
posted by: Tumor Research
[Abstract]
This research was composed of two parts:Ⅰ. Establishment of a novel assay for detecting mutations associated with antiviral resistance of HBV covalently closed circular DNA.Ⅱ. Detection and comparison of mutations associated with antiviral resistance of cccDNA in liver cells and HBV DNA in peripheral serum of patients chronically infected with hepatitis B virus.Ⅰ. Establishment of a novel assay for detecting mutations associated with antiviral resistance of HBV covalently closed circular DNAObjective To establish an assay for detecting mutations associated with antiviral resistance of hepatitis B virus covalently closed circular DNA(HBV cccDNA). Methods①A selective Polymerase chain reaction (PCR) system based on primers set spanning the two gaps(DR regions) in HBV genome was established to preferentially amplify the region encoding lamivudine, adefovir and enticavir resistant substitutions of HBV cccDNA. To improve the sensivity of PCR, the PCR conditions such as the annealing temperature, the concentration of Mg2+ and Dimethyl sulfoxide(DMSO) were optimized. And the efficiency of this system to discriminate cccDNA and rcDNA was verified with HBV DNA molecules in peripheral serum and purified plasmid containing whole HBV DNA sequence (cccDNA).②The co-precipitation of Sodium Dodecyl Sulfate (SDS) and protein combining with the QIAamp DNA Mini Kit(tissue) were used to selectively extract HBV cccDNA. The extracted products were further purified with plasmid-safe ATP-dependent DNase(PSAD). HBV DNA and cccDNA in both supernatant and sediment were quantified using Real-time quantitative Polymerase Chain Reaction (Real Time PCR), and the impact of proteinase K on extracting efficiency was evaluated. ③The impact of DMSO on PCR was tested by comparison of the DNA sequencing results of the routine PCR with standard buffer and the nested PCR with buffer containing DMSO. Results①In this novel PCR system, the optimized concentration of Mg2+ and DMSO were 2mM and 2%. No detectable fluorescent signal was observed when the HBV DNA in peripheral serum samples were about 5x 106 copies per millilitre. The detectable HBV cccDNA was as low as 103 copies per millilitre at least by this method.②HBV DNA in peripheral serum was declined about 21og10 post hydrolyzed, and no impact of PS AD on cccDNA was detected.③When using co-precipitation of SDS and protein combining with the QIAamp DNA Mini Kit, the absence of proteinase K in digestive juice improved the yield of HBV rcDNA in the sedimentation, therefore raised the differentiation between rcDNA and cccDNA. And it didn’t impact the yield of HBV cccDNA in the superstratum.④The DNA sequencing results of nested PCR with buffer containing DMSO was consistent with the routine PCR with standard buffer. Conclusions Selective extraction of HBV cccDNA based on the co-precipitation of SDS-protein and the QIAamp DNA Mini Kit, and further purification with PSAD, integrating with selective PCR system can ensure the specific amplification of HBV cccDNA. The nested PCR with buffer containing 2% DMSO has no impact on DNA sequencing results. This method can be utilized in detection of mutations associated with antiviral resistance of intracellular HBV cccDNA. It is convenient, highly specific and highly sensitive.Ⅱ. Detection and comparison of mutations associated with antiviral resistance of cccDNA in liver cells and HBV DNA in peripheral serum of patients chronically infected with hepatitis B virus.Objective To determine the existence of mutations associated with antiviral resistance of cccDNA in liver cells of chronic HBV infected patients, and study the differences of virus strains in the liver cells and peripheral. Methods①Forty patients chronically infected with hepatitis B virus were investigated. Serum before operation and non-tumor liver tissues were selected.②The serum HBV DNA was extracted by directly boiling.③The HBV cccDNA and rcDNA were selectively extracted by co-precipitation using SDS-protein and the QIAamp DNA Mini Kit, and further purification with PSAD. To assess the effect of extraction and purification, the whole products were detected by real-time fluorescent quantitative PCR.④All the samples were amplified by PCR or nested PCR with the corresponding primers, and then the positive PCR products were sequenced commercialy. Results①Among the whole 40 patients, HBV DNA were detected in serum in 21 patients, HBV cccDNA were positive in liver cells in 31 patients, and HBV rcDNA were positive in liver cells in 37 patients. DNA sequences of these samples were successfully tested.②Mutations of rtM204I were detected in intracellular HBV cccDNA, rcDNA and serum HBV DNA of 2 patients. Mutations of rtM204I, rtQ215H were detected in intracellular HBV cccDNA and rcDNA of 2 patient, while the corresponding mutation was not detected in serum HBV DNA. Mutations of rtM204V, rtM204V, rtV173L+rtL180M+rtM204V were detected respectively in serum HBV DNA in 3 patients, while the corresponding mutants were not detected in intracellular HBV cccDNA or rcDNA. There also existed some other mutants on the various HBV cccDNA base sites. Conclusions Our results suggested that there are mutations associated with antiviral resistance of intracellular hepatitis B virus cccDNA in patients chronically infected with HBV. And the dominant virus quasispecies are different between HBV in liver cells and peripheral serum. There are more than one templates of HBV in liver cells.
Title: Establishment of a Novel Assay for Detecting Mutations Associated with Antiviral Resistance of HBV Covalently Closed Circular DNA and Its Application
i guess the acute flares are due to the mutants escaping immune pressure, and this does happen even with low hbvdna, of course the low hbvdna may take decades to make the mutants
One thing that often bugs me: the same mutation must occur in many different infected liver cells before the mutation exerts any influence. I can understand if there is selection pressure due to drugs or immune control. Without selection pressure, the mutants must compete with each other and the wild type. How the hell do we get acute flares now and then?
I'd think any level grater than the level of the wild type would make them a dominant level and at any level they will be like latent time bombs in waiting to go offeven if clinically inactive.
Just my 2 cents
Just my 2 cents
A good one. I wonder what levels mutants must reach before they become dominant or can they exert influence clinically even at low concentrations?
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