| Hepatitis B virus (HBV) infection is one of the most common viral diseases severely threatening human health. China is one of the regions where HBV is prevalent most in the world. There are about 10% people carried with HBsAg, and 30 million chronic hepatitis B (CHB). Without effective rigimon and medicines, 300 thousand of patients died of serious hepatitis, liver cirrhosis and hepatocellular carcinoma caused by HBV infection. It has become a most important public health problem to prevent and cure HBV-related diseases.Clinical and experimental studies indicate that although the HBV markers in their serum is negative the symptoms of some HBV patients do not been improved and viral replication does not stop. Recent years it has been recognized that the failure to eliminate cccDNA thoroughly is the radical reason.After the genome of HBV entering the cellular nucleus, the rcDNA (relaxed circular DNA) is converted to cccDNA (covalently closed circular DNA), which is the original template serving as a matrix for mRNA transcription. Persistence of cccDNA in hepatocyte plays a key role in the long term infection of HBV, and it has become the most important indication of HBV infection. Although the antiviral drugs can clean the HBV and reduce the level of HBV-DNA in peripheral blood, they can not prevent the restoration of cccDNA pool effectively. HBVcccDNA is the most valuable index for HBV replication, viral infectivity and efficacy of antiviral therapy. Only HBVcccDNA is been clean the carrier state of HBV patients can be avoided. Therefore, to establish a specific and sensitive method for the detection of cccDNA will be an important task for HBV research, clinical diagnosis, condition judgment and therapeutic evaluation.On account of those mentioned above, the purpose of our study is to establish a specific and sensitive method for quantitive detection of HBVcccDNA, thereafter try to use the method to detect the HBVcccDNA from the PBMCs or sera of CHB patients.1. The preparation of standard for HBV-cccDNA detection Plasmid pBR322ADR, named as P1.2Ⅱ, which includes whole genome of adr-type HBV and has no gaps near direct repeat sequence, is served as standard preparation for HBVcccDNA detection. P1.2Ⅱhas intact duplex structure similar to HBVcccDNA. After amplification of P1.2Ⅱ, quantitation is performed 3 times with ultraviolet spectrophotometer, and the mean of reading is used in formula described below: Copies= (M/W)×6.02×1023. Proper dilution will be performed when necessary.2. The establishment of fluorescent quantitative real-time PCR for HBV-cccDNA detectionAccording to the difference of structure and physico-chemical property between HBV genome and cccDNA, a pair of specific primers spanning the gap of double strand were designed and synthesized. At the same time, a specific TaqMan probe locating the vicinity of the"gap"and downstream of negative strand was also constructed Then amplification of specific HBV fragment (≈365bp) was performed using Fluorescent Quantitative Machine. Plasmid purification kit which can remove rcDNA was used to obtain template DNA. The DNA were digested and purified by Plasmid-SafeTM ATP-Dependent DNase (PSAD) to digest rcDNA further3. The establishment of nested-real time fluorescent quantitative PCR for HBV-cccDNA detectionThe sensitivity of fluorescent quantitation is often affected by the size of DNA segment amplified and it has been known that the optimal size is 50bp to 150bp. Since the sensitivity of fluorescent quantitative PCR is not satisfied the nested-fluorescent quantitative real time PCR was established. A pair of outer primers locating at the sides of the gap of the double strains and inner primers locating at that of the negative strain was designed to amplify the specific segment. The TaqMan probe was also used in this course. After normal amplification with outer primers, products were used for real time PCR with inner primers and fluorescent probe. Clearance of rcDNA was performed as mentioned before. Then the original copy of HBVcccDNA was measured with standard preparation as reference.4. Quantitive detection of HBVcccDNA in PBMC or sera from CHB patients The PBMC and sera of 34 cases of CHB patients, sera of 14 carriers and sera of 12 cases of health examination were detected for HBVcccDNA as described before. After DNA extraction in PBMC or sera using plasmids extracted kits or QIAamp MinElute Virus Spin Kit, respectively, the templates were purified and digested with PSAD. Then nested-real time PCR amplification was performed. Statistic analysis was used for the data from measurement of ALT, HBV DNA and cccDNA in sera or PBMC from 34 CHB patients.Results1. Real time PCR is not suitable for HBVcccDNA detection because the sensitivity is not so high and the lower limit is1×105 copies/mL.2. The lower limit of nested-real time PCR is 1×102 copies/mL. Products of positive amplification have no basi-depletion or mutation identified by gene sequencing. Sequence homology of products reached to 98%~100% comparing with other strains sequences in Genbank.3. HBV-DNA in PBMC or sera of 34 CHB patients is positive. The cccDNA was detected in PBMC of 28 cases (82.35%) and in the sera of 25 cases (73.53%).4. The cccDNA was detected in sera of 6 carriers (42.86%) and in sera of 2 cases of health examination accompany with HBsAb positive.5. Significant positive correlations were shown among cccDNA in sera, DNA in sera and DNA in PBMC. There were also significant correlations between HBsAg and cccDNA in sera.Conclusions1. Nested-real time PCR is specific and sensitive for HBVcccDNA detection in PBMC or sera.2. Contents of cccDNA in PBMC or sera are low. PBMC may serve as reservoir for cccDNA, and the cccDNA detection in PBMC can be used to evaluate the antiviral effect, to decide the end point of antiviral therapy, and to predict relapse of illness.3. HBVcccDNA detected in sera can partlly reflects the level of HBVcccDNA in hepatic tissue. It can be used as an early signal of the activation of HBV and the liver damage in CHB patients .
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