Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA / 中华医学杂志(英文版)
Chinese Medical Journal
; (24): 1513-1516, 2011.
Article
in English
| WPRIM (Western Pacific)
| ID: wpr-353953
Responsible library:
WPRO
ABSTRACT
<p><b>BACKGROUND</b>Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus (HBV) DNA replication, the covalently closed circular HBV DNA (cccDNA) can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction (PCR) assay for detecting HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs).</p><p><b>METHODS</b>Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA (rcDNA), two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard.</p><p><b>RESULTS</b>The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0 × 10(2) copies/ml to 3.9 × 10(8) copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative.</p><p><b>CONCLUSION</b>The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.</p>
Full text:
Available
Health context:
SDG3 - Health and Well-Being
Health problem:
Target 3.3: End transmission of communicable diseases
Database:
WPRIM (Western Pacific)
Main subject:
DNA, Circular
/
DNA, Viral
/
Cells, Cultured
/
Hepatitis B virus
/
Real-Time Polymerase Chain Reaction
/
Genetics
/
Methods
Type of study:
Diagnostic study
Limits:
Humans
Language:
English
Journal:
Chinese Medical Journal
Year:
2011
Document type:
Article