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
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
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DOI：
10.1007/978-1-61779-937-2_7
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发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
Beld, M. G. H. M.
中科院分区：
文献类型：
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作者：
Takkenberg, R. B.;Menting, S.;Beld, M. G. H. M.
B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感染的持续性中发挥作用。然而，cccDNA在血浆中的起源和意义还不清楚。开发并验证了用于检测和定量慢性B型肝炎患者血浆中cccDNA的灵敏、特异和可重复的实时PCR。本文分析了4种HBV DNA参考品和96份慢性B型肝炎患者血浆标本。结果与总HBV DNA水平进行比较。该cccDNA测定法的检测下限为15个拷贝/PCR，定量下限为91个拷贝/PCR，相关系数（R）为0.98（p < 0.0001）。HBV cccDNA可在四个国际样本组中的两个中检出。在两组（R = 0.96和R = 0.43）和慢性B型肝炎患者样本（R = 0.88，p < 0.0001）中发现cccDNA和总HBV DNA水平之间存在显著相关性。在这些样品的57%中可以检测到cccDNA。cccDNA的平均水平为总HBV载量的0.16%。HBeAg阳性样本的血浆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。
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.