Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA.
Chin Med J (Engl)
; 124(10): 1513-6, 2011 May.
Article
en En
| MEDLINE
| ID: mdl-21740808
ABSTRACT
BACKGROUND:
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).METHODS:
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.RESULTS:
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.CONCLUSION:
The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.
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Colección:
01-internacional
Base de datos:
MEDLINE
Contexto en salud:
2_ODS3
Problema de salud:
2_enfermedades_transmissibles
Asunto principal:
ADN Circular
/
ADN Viral
/
Virus de la Hepatitis B
/
Reacción en Cadena en Tiempo Real de la Polimerasa
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
Chin Med J (Engl)
Año:
2011
Tipo del documento:
Article
País de afiliación:
China