title: Quantitation of hepatitis delta virus using a single-step internally controlled real-time RT-qPCR and a full-length genomic RNA calibration standard.
abstract: Quantitation of circulating hepatitis delta virus (HDV) RNA is important for assessing the response to antiviral therapy and for understanding the complex dynamic interactions between hepatitis B virus (HBV) and HDV replication. Although several PCR assays for HDV RNA have been described none of them incorporate an internal control or use a full-length RNA calibration standard for absolute quantitation. This study describes the development and evaluation of a novel single-step real-time RT-qPCR assay for HDV RNA quantitation which incorporates a Brome Mosaic virus internal control to prevent false negatives and under-reporting due to inhibitors or due to inefficient RNA purification, reverse transcription or PCR amplification. The assay has a dynamic range of ≥7log(10) and is designed to detect all HDV genotypes. The 95% detection limit is ∼3800 HDV RNA copies/ml, 700 copies/ml being detectable in 20% of repeats. Both intra-assay and inter-assay variability are low (CV 8% and 17%, respectively). Plasma HDV RNA was detected in 75% of 59 HDV antibody-positive samples with titres ranging from 8.4×10(4) to 4.4×10(8) copies/ml. The assay described provides a reliable and sensitive quantitative system for therapeutic monitoring and for studying the dynamic interplay between hepatitis B virus replication and HDV viral load.
authors: Research Department of Infection, Division of Infection and Immunity, University College London, London, UK. r.ferns@ucl.ac.uk
authors: R B
authors: RB
authors: Ferns
authors: E
authors: E
authors: Nastouli
authors: J A
authors: JA
authors: Garson
mesh_terms: D017795:Bromovirus / Q000235:genetics; D003698:Hepatitis Delta Virus / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods* / Q000592:standards
chemical_list: D012367:RNA, Viral