Establishment and application of nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA
10.3760/cma.j.issn.1003-9279.2011.04.023
- VernacularTitle:单核细胞乙型肝炎病毒cccDNA检测方法及应用价值
- Author:
Chun-Hai XU
1
;
Zhao-Shen LI
;
Jun-Ying DAI
;
Hai-Yang ZHU
;
Jian-Wu YU
;
Shu-Lan LV
Author Information
1. 哈尔滨医科大学第二附属医院
- Keywords:
Hepatitis B virus;
DNA;
Polymerasc chain reaction;
Monocytes
- From:
Chinese Journal of Experimental and Clinical Virology
2011;25(4):307-309
- CountryChina
- Language:Chinese
-
Abstract:
Objective 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). MethodsBased 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. Results We have established a nested real-time fluorescent quantitative PCR method for HBV cccDNA successfully,and the linear range is from 5.0 × 102 to 3. 9 × 107 copies per milliliter. Of the 25 PBMC samples and 7MMNC 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.ConclusionsThe 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.
