Establishment and application of nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA / 中华实验和临床病毒学杂志
Chinese Journal of Experimental and Clinical Virology
; (6): 307-309, 2011.
Article
in Zh
| WPRIM
| ID: wpr-246256
Responsible library:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To establish a nested real-time quantitative polymerase chain reaction (PCR) assay for detection of hepatitis B virus covalently closed circular DNA in PBMC( peripheral blood monocyte) and MMNC (marrow monocyte).</p><p><b>METHODS</b>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. To remove rcDNA, cccDNA was processed by Mung Bean Nuclease,and then 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.</p><p><b>RESULTS</b>We have established a nested real-time fluorescent quantitative PCR method for HBV cccDNA successfully, and the linear range is from 5.0 x 10(2) to 3. 9 x 10(7) copies per milliliter. Of the 25 PBMC samples and 7 MMNC samples of the chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were HBV cccDNA positive, while all of the 21 healthy donator blood PBMC samples were negative.</p><p><b>CONCLUSIONS</b>The nested real-time fluorescent quantitative PCR method may be applied to detect HBVcccDNA level in PBMC and MMNC. HBVcccDNA can be detected in PBMC and MMNC.</p>
Full text:
1
Index:
WPRIM
Main subject:
Virology
/
DNA, Circular
/
DNA, Viral
/
Leukocytes, Mononuclear
/
Hepatitis B virus
/
Sensitivity and Specificity
/
Diagnosis
/
Real-Time Polymerase Chain Reaction
/
Genetics
/
Hepatitis B
Type of study:
Diagnostic_studies
Limits:
Humans
Language:
Zh
Journal:
Chinese Journal of Experimental and Clinical Virology
Year:
2011
Type:
Article