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
de En
| WPRIM
| ID: wpr-353953
Bibliothèque responsable:
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>
Texte intégral:
1
Indice:
WPRIM
Sujet Principal:
ADN circulaire
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ADN viral
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Cellules cultivées
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Virus de l'hépatite B
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Réaction de polymérisation en chaine en temps réel
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Génétique
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Méthodes
Type d'étude:
Diagnostic_studies
Limites du sujet:
Humans
langue:
En
Texte intégral:
Chinese Medical Journal
Année:
2011
Type:
Article