OBJECTIVE: To establish a nested real-time quantitative polymerase chain reaction (PCR) assay for detection of hepatitis B virus covalently closed circular DNA in PBMC( peripheral blood monocyte) and MMNC (marrow monocyte). METHODS: Based on the structural differences between HBVcccDNA and HBV rcDNA, two pairs of specific primers spanned the gap of the positive and negative chains and a specific TaqMan probe situated downstream were designed. To remove rcDNA, cccDNA was processed by Mung Bean Nuclease,and then amplified by nested real-time quantitative PCR using a pair of outer primers and a pair of inner primers. According to the standard preparation, cccDNA levels of specimen were calculated. RESULTS: We have established a nested real-time fluorescent quantitative PCR method for HBV cccDNA successfully, and the linear range is from 5.0 x 10(2) to 3. 9 x 10(7) copies per milliliter. Of the 25 PBMC samples and 7 MMNC samples of the chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were HBV cccDNA positive, while all of the 21 healthy donator blood PBMC samples were negative. CONCLUSIONS: The nested real-time fluorescent quantitative PCR method may be applied to detect HBVcccDNA level in PBMC and MMNC. HBVcccDNA can be detected in PBMC and MMNC.
OBJECTIVE: To establish a nested real-time quantitative polymerase chain reaction (PCR) assay for detection of hepatitis B virus covalently closed circular DNA in PBMC( peripheral blood monocyte) and MMNC (marrow monocyte). METHODS: Based on the structural differences between HBVcccDNA and HBV rcDNA, two pairs of specific primers spanned the gap of the positive and negative chains and a specific TaqMan probe situated downstream were designed. To remove rcDNA, cccDNA was processed by Mung Bean Nuclease,and then amplified by nested real-time quantitative PCR using a pair of outer primers and a pair of inner primers. According to the standard preparation, cccDNA levels of specimen were calculated. RESULTS: We have established a nested real-time fluorescent quantitative PCR method for HBV cccDNA successfully, and the linear range is from 5.0 x 10(2) to 3. 9 x 10(7) copies per milliliter. Of the 25 PBMC samples and 7 MMNC samples of the chronic hepatitis B or liver cirrhosispatients, 3 MMNC samples and 9 PBMC samples were HBV cccDNA positive, while all of the 21 healthy donator blood PBMC samples were negative. CONCLUSIONS: The nested real-time fluorescent quantitative PCR method may be applied to detect HBVcccDNA level in PBMC and MMNC. HBVcccDNA can be detected in PBMC and MMNC.