title: Quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in the liver of HBV-infected patients by LightCycler real-time PCR.
abstract: Antivirals for hepatitis B virus (HBV) reduce viral load and improve liver histology, however, their effect on covalently closed circular DNA (cccDNA), the HBV transcriptional template, has not been extensively examined. This study evaluated a newly designed LightCycler based quantitative cccDNA PCR assay. A linear range of 2.5 x 10(1) to 1 x 10(9) copies/assay using primers specific for HBV cccDNA and 2.5 x 10(1) to 2.5 x 10(9) copies/assay using primers specific for total HBV DNA (tDNA) was established. beta-Globin was used to estimate the number of cells in each PCR reaction. Enzymatic digestion with an ATP-dependent DNase improved the analytic specificity to a greater than 1:10000 ratio of cccDNA:RC DNA (relaxed circular DNA). One-tenth of the extracted DNA from 1mg of liver biopsy, was analyzed from six patients, three HBV-infected and three uninfected individuals, under blinded conditions; three were found positive and three negative for cccDNA and tDNA. Approximately 6 x 10(3) copies of cccDNA/mg of tissue were detected in a pre-transplant biopsy from an HBV-infected patient treated with lamivudine. Sequential post-transplant liver biopsies were negative for both HBV cccDNA and tDNA. An HBV-infected patient with cirrhosis who was antiviral therapy naïve had 3.7 x 10(4) copies of cccDNA/mg of liver tissue. Another treatment-naïve patient with a history of high HBV viral load had 1 x 10(5) copies of cccDNA/mg of tissue (4 x 10 (6) copies of tDNA/mg of tissue). Further studies are warranted but the high level of sensitivity, specificity, rapidity and accuracy provided by this novel assay with the LightCycler system indicate that it could be useful for monitoring antiviral therapy.
authors.affiliation: Viridae Clinical Sciences Inc., 1134 Burrard Street, Vancouver, BC, Canada V6Z 1Y8.
authors.forename: Madhu
authors.initials: M
authors.lastname: Singh
authors.forename: Annie
authors.initials: A
authors.lastname: Dicaire
authors.forename: Adil E
authors.initials: AE
authors.lastname: Wakil
authors.forename: Carolyn
authors.initials: C
authors.lastname: Luscombe
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Sacks
mesh_terms: D004270:DNA, Circular / Q000032:analysis* / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D019694:Hepatitis B, Chronic / Q000821:virology; D006801:Humans; D008099:Liver / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D019914:Taq Polymerase