Development and clinical applications of improved assays for detection and quantitation of hepatitis C virus
Development and clinical applications of improved assays for detection and quantitation of hepatitis C virus
For this project, a real-time PCR-based assay was developed for detection and quantitation of HCV RNA using the TaqMan 5' nuclease assay and the Applied Biosystems PRISM 7700 Sequence Detection System.
TaqMan primers and probes were designed to amplify part of the 5' untranslated region and detected HCV genotypes 1 - 5 with equal efficiency. An HCV standard RNA was developed and used to generate standard curves for quantitation. An internal control RNA was produced and used to monitor efficiency of RNA extraction reverse transcription and amplification.
The performance of the assay was evaluated using the World Health Organisation International Standard for HCV RNA. The assay quantified the International Standard to within a mean of 27% from the published load and had a limit of detection of 50 international units/ml. The assay had a wide dynamic range (50 - 108 copies/ml) and had intra and inter-assay coefficients of variation of 23.8% and 29.4%, respectively. The assay was compared with the Quantiplex HCV RNA 2.0 assay (Bayer) and viral loads given by the two assays correlated strongly.
The quantitative assay was used to study HCV load in 53 symptomatic and 57 asymptomatic chronically infected individuals. No difference in load was found between the two populations. When the groups were combined, a weak correlation was found between viral load and the level of liver fibrosis. Longitudinal variation in load was also studied in 19 asymptomatic patients. Of these, eight showed an increase in load, ten showed a decrease and one showed no overall change in load. Viral load varied by <11og10 copies/ml in the majority of patients (68%).
HCV loads were also studied in eight HCV-positive patients treated with a combination of pegylated interferon and ribavirin (Roche). End of treatment virological and biochemical responses rates were 87.5% and 78%, respectively and the overall end of treatment response rate was 75%. Highest rates of decline in viral load were seen in patients infected with genotype 3 virus.
The assay was also used to quantify HCV RNA in cultured peripheral blood mononuclear cells from HCV-positive patients. The cells were treated with immunomodulatory compounds (cyclosporin A, hydrocortisone, phytohaemagglutinin) and HCV RNA levels measured. HCV RNA levels decreased in all treated and untreated cells over the study period (seven to 14 days). The addition of the immunomodulatory compounds had no detectable effects on virus levels.
University of Southampton
Hadfield, Stephen J
9cf298cd-f8ba-4eb8-9f9a-aaf3b615e7fc
2002
Hadfield, Stephen J
9cf298cd-f8ba-4eb8-9f9a-aaf3b615e7fc
Hadfield, Stephen J
(2002)
Development and clinical applications of improved assays for detection and quantitation of hepatitis C virus.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
For this project, a real-time PCR-based assay was developed for detection and quantitation of HCV RNA using the TaqMan 5' nuclease assay and the Applied Biosystems PRISM 7700 Sequence Detection System.
TaqMan primers and probes were designed to amplify part of the 5' untranslated region and detected HCV genotypes 1 - 5 with equal efficiency. An HCV standard RNA was developed and used to generate standard curves for quantitation. An internal control RNA was produced and used to monitor efficiency of RNA extraction reverse transcription and amplification.
The performance of the assay was evaluated using the World Health Organisation International Standard for HCV RNA. The assay quantified the International Standard to within a mean of 27% from the published load and had a limit of detection of 50 international units/ml. The assay had a wide dynamic range (50 - 108 copies/ml) and had intra and inter-assay coefficients of variation of 23.8% and 29.4%, respectively. The assay was compared with the Quantiplex HCV RNA 2.0 assay (Bayer) and viral loads given by the two assays correlated strongly.
The quantitative assay was used to study HCV load in 53 symptomatic and 57 asymptomatic chronically infected individuals. No difference in load was found between the two populations. When the groups were combined, a weak correlation was found between viral load and the level of liver fibrosis. Longitudinal variation in load was also studied in 19 asymptomatic patients. Of these, eight showed an increase in load, ten showed a decrease and one showed no overall change in load. Viral load varied by <11og10 copies/ml in the majority of patients (68%).
HCV loads were also studied in eight HCV-positive patients treated with a combination of pegylated interferon and ribavirin (Roche). End of treatment virological and biochemical responses rates were 87.5% and 78%, respectively and the overall end of treatment response rate was 75%. Highest rates of decline in viral load were seen in patients infected with genotype 3 virus.
The assay was also used to quantify HCV RNA in cultured peripheral blood mononuclear cells from HCV-positive patients. The cells were treated with immunomodulatory compounds (cyclosporin A, hydrocortisone, phytohaemagglutinin) and HCV RNA levels measured. HCV RNA levels decreased in all treated and untreated cells over the study period (seven to 14 days). The addition of the immunomodulatory compounds had no detectable effects on virus levels.
Text
842824.pdf
- Version of Record
More information
Published date: 2002
Identifiers
Local EPrints ID: 464700
URI: http://eprints.soton.ac.uk/id/eprint/464700
PURE UUID: 48a74a61-c473-4ea3-b5d7-8a98b0461428
Catalogue record
Date deposited: 04 Jul 2022 23:57
Last modified: 16 Mar 2024 19:42
Export record
Contributors
Author:
Stephen J Hadfield
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics