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Requirement of cyclin-dependent kinase function for hepatitis B virus cccDNA synthesis as measured by digital PCR.
Bao, Ching-Yu; Hung, Hsu-Chin; Chen, Yi-Wen; Fan, Chiao-Yuan; Huang, Chien-Jung; Huang, Wenya.
Afiliação
  • Bao CY; Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
  • Hung HC; Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
  • Chen YW; Cold Spring Biotech Corp, New Taipei City, Taiwan.
  • Fan CY; Cold Spring Biotech Corp, New Taipei City, Taiwan.
  • Huang CJ; Department of Internal Medicine, Taipei City Hospital, Taipei, Taiwan.
  • Huang W; Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Institute of Basic Medicine, National Cheng Kung University, Tainan, Taiwan; Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan. Electronic a
Ann Hepatol ; 19(3): 280-286, 2020.
Article em En | MEDLINE | ID: mdl-31964596
INTRODUCTION AND OBJECTIVES: HBV covalently closed circular (ccc) DNA is the key player in viral persistence and an important predictive biomarker for hepatitis relapse. Precise quantification of intracellular cccDNA is challenging because cccDNA is present in very low levels in hepatocytes, where it also co-exists with a large excess amount of relaxed circular (rc) DNA. We aimed to develop a highly sensitive cccDNA detection method for cccDNA quantification by digital PCR (dPCR). PATIENTS OR MATERIALS AND METHODS: A standard plasmid containing the whole HBV genome in the closed circular conformation was employed to characterize the performance of dPCR. rcDNA in the growth medium of HBV-producing HepAD38 cells was used as a matrix for cccDNA detection. Intrahepatic cccDNA measurement by dPCR and qPCR was performed to determine the correlation of the analysis results for the two methods. RESULTS: The limit of detection (LOD) of the cccDNA dPCR was 1.05copy/µl, and the linear range of detection was 1.02×104copies/µl, achieving a dynamic detection range of 104-fold. cccDNA measurement using excess rcDNA as the matrix did not reveal false-positive detection, indicating that dPCR was highly specific. In the HepAD38 cells, the cccDNA levels measured by dPCR were highly correlated with those measured by qPCR but had a higher sensitivity. The CDK inhibitor AZD-5438 was found to block intracellular cccDNA synthesis. CONCLUSIONS: Dpcr greatly improved the sensitivity and specificity of cccDNA detection. Host CDK activities are likely required for cccDNA synthesis. dPCR can potentially be applied for drug screening for effective cccDNA inhibitors.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: DNA Circular / DNA Viral / Vírus da Hepatite B / Reação em Cadeia da Polimerase / Quinases Ciclina-Dependentes / Hepatócitos Limite: Humans Idioma: En Revista: Ann Hepatol Assunto da revista: GASTROENTEROLOGIA Ano de publicação: 2020 Tipo de documento: Article País de afiliação: Taiwan País de publicação: México
Texto completo
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: DNA Circular / DNA Viral / Vírus da Hepatite B / Reação em Cadeia da Polimerase / Quinases Ciclina-Dependentes / Hepatócitos Limite: Humans Idioma: En Revista: Ann Hepatol Assunto da revista: GASTROENTEROLOGIA Ano de publicação: 2020 Tipo de documento: Article País de afiliação: Taiwan País de publicação: México