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ISSN 1001-5256 (Print)
ISSN 2097-3497 (Online)
CN 22-1108/R
Issue 3
Mar.  2011
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Article Contents

Establishment and application of nested real-time fluorescent quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA

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  • Published Date: 2011-03-20
  • Objective To establish a nested real-time quantitative polymerase chain reaction (PCR) assay for detection of hepatitis B virus covalently closed circular DNA (cccDNA) in peripheral blood monocyte (PBMC) and marrow monocyte (MMNC) respectively.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.RcDNA was degraded and cccDNA was processed by Mung Bean Nuclease, cccDNA was 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 established a nested real-time fluorescent quantitative PCR method for HBV cccDNA successfully, and the linear range is from 3.0×102 to 3.9×l08 copies per milliliter.Of the 25 PBMC samples and 7 MMNC samples of the chronic hepatitis B or liver cirrhosis patients, 9 PBMC samples and 3 MMNC samples were HBV cccDNA positive, while all of the 21 healthy donator blood PBMC samples were negative.Conclusion The nested real-time fluorescent quantitative PCR method may be applied to detect HBV cccDNA in PBMC and MMNC of patients with HBV.HBV cccDNA can be detected in PBMC and MMNC.

     

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