title: Development and validation of an efficient in-house real-time reverse transcription polymerase chain reaction assay for the quantitative detection of serum hepatitis delta virus RNA in a diverse South London population.
abstract: Hepatitis delta virus (HDV) causes both acute and chronic hepatitis, always in the presence of hepatitis B. Analysis of global HDV isolates has shown that at least eight genotypes exist. HDV RNA quantitation and genotyping are important tools in the diagnosis and management of infected individuals. There is, as yet, no commercially available quantitative HDV RNA assay. Several laboratories have developed in-house assays, but equivalent detection and quantitation across all HDV genotypes has not been demonstrated. In this study, the development of an in-house real-time reverse transcription polymerase chain reaction (RT PCR) assay is described to quantify HDV RNA in serum or plasma. Its efficiency was validated by testing 99 samples from patients with known chronic HDV infection, along with 22 samples from individuals without HDV. The assay has a dynamic range of 6.4×10(2) to 6.4×10(8)copies/mL. Amplicons of the quantitative PCR can be directly used for sequence analysis and genotyping. HDV-1, HDV-5 and HDV-6 were identified, reflecting the areas of origin of our cohort of patients. The ability to genotype and to accurately quantify HDV RNA levels in the more recently discovered African genotypes will be important for investigating the natural history of HDV in this group, compared to those with genotype 1 disease.
authors.affiliation: Institute of Liver Studies, King's College Hospital, Denmark Hill, London, SE5 9RS, UK.
authors.forename: Dazhuang
authors.initials: D
authors.lastname: Shang
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Hughes
authors.forename: Mary
authors.initials: M
authors.lastname: Horner
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Bruce
authors.forename: Yan
authors.initials: Y
authors.lastname: Dong
authors.forename: Ivana
authors.initials: I
authors.lastname: Carey
authors.forename: Abid R
authors.initials: AR
authors.lastname: Suddle
authors.forename: Kosh
authors.initials: K
authors.lastname: Agarwal
authors.forename: Phillip M
authors.initials: PM
authors.lastname: Harrison
authors.forename: Mark
authors.initials: M
authors.lastname: Atkins
mesh_terms: D003699:Hepatitis D / Q000821:virology*; D003698:Hepatitis Delta Virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008131:London; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D044967:Serum / Q000821:virology*; D019562:Viral Load / Q000379:methods*
chemical_list: D012367:RNA, Viral