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.
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.
Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
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RB
Takkenberg
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S
Menting
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MG
Beld
D004270:DNA, Circular / Q000097:blood* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004279:DNA, Viral / Q000097:blood* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005838:Genotype; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D019694:Hepatitis B, Chronic / Q000097:blood*; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D025202:Molecular Diagnostic Techniques; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results
D004270:DNA, Circular; D004279:DNA, Viral