一种用于检测和定量慢性乙型肝炎患者血浆中乙型肝炎病毒共价闭合环状DNA的灵敏且特异的实时聚合酶链反应的验证

Validation of a sensitive and specific real-time PCR for detection and quantitation of hepatitis B virus covalently closed circular DNA in plasma of chronic hepatitis B patients.

作者信息

Takkenberg R B, Menting S, Beld M G H M

机构信息

Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

出版信息

Methods Mol Biol. 2012;903:113-28. doi: 10.1007/978-1-61779-937-2_7.

DOI:10.1007/978-1-61779-937-2_7

PMID:22782814

Abstract

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) serves as a template for viral replication and plays a role in persistence of HBV infection. The origin and significance of cccDNA in plasma, however, are not well understood. A sensitive, specific, and reproducible real-time PCR for detection and quantitation of cccDNA in plasma of chronic hepatitis B patients was developed and validated. Four HBV DNA reference panels and 96 plasma samples of chronic hepatitis B patients are analyzed. Results are compared with total HBV DNA levels. This cccDNA assay had a lower limit of detection at 15 copies/PCR, a lower limit of quantitation at 91 copies/PCR, and a correlation coefficient (R) of 0.98 (p < 0.0001). HBV cccDNA can be detected in two of four international panels. Significant correlation is found between cccDNA and total HBV DNA levels in both panels (R = 0.96 and R = 0.43) and in samples of the chronic hepatitis B patients (R = 0.88, p < 0.0001). In 57 % of these samples cccDNA can be detected. Mean level of cccDNA is 0.16 % of total HBV load. Plasma HBV cccDNA levels are higher in HBeAg-positive samples than in HBeAg-negative samples (p < 0.0001). Total HBV DNA levels and HBV genotype do not influence cccDNA detection.

摘要

乙型肝炎病毒（HBV）共价闭合环状DNA（cccDNA）作为病毒复制的模板，在HBV感染的持续存在中发挥作用。然而，血浆中cccDNA的来源和意义尚未完全明确。我们开发并验证了一种用于检测和定量慢性乙型肝炎患者血浆中cccDNA的灵敏、特异且可重复的实时PCR方法。分析了四个HBV DNA参考品组和96份慢性乙型肝炎患者的血浆样本。将结果与总HBV DNA水平进行比较。该cccDNA检测方法的检测下限为15拷贝/PCR，定量下限为91拷贝/PCR，相关系数（R）为0.98（p<0.0001）。在四个国际品组中的两个中可检测到HBV cccDNA。在两个品组以及慢性乙型肝炎患者样本中，cccDNA与总HBV DNA水平之间均存在显著相关性（R分别为0.96和0.43，以及R = 0.88，p<0.0001）。在这些样本中，57%可检测到cccDNA。cccDNA的平均水平为总HBV载量的0.16%。HBeAg阳性样本中的血浆HBV cccDNA水平高于HBeAg阴性样本（p<0.0001）。总HBV DNA水平和HBV基因型不影响cccDNA检测。