Screening for genetic heterogeneity in the interferon sensitivity determining region of the hepatitis C virus genome by polymerase chain reaction with melting curve analysis
-
Daisuke Sasaki
Abstract
Background: Although mutations in the interferon (IFN) sensitivity determining region (ISDR) of hepatitis C virus (HCV) have been reported to be useful as a predictive viral factor for IFN therapy in patients infected with HCV-1b, such laboratory research has not been favorably translated into the clinic. To promote such translation, we attempted the establishment of a rapid and simple polymerase chain reaction (PCR) combined with melting curve analysis (MCA) to screen for mutations in the ISDR and for the monitoring of HCV quasispecies.
Methods: A PCR-MCA protocol was established using in-house primers and hybridization probes designed according to the results of direct sequencing of 34 HCV-1b samples. Then, the performance of PCR-MCA was verified by comparing with mutation profiles obtained by direct sequencing and sequencing after cloning.
Results: The MCA assay revealed that melting temperature (Tm) was inversely correlated with the number of nucleotide (nt) and amino acid substitutions in the ISDR deduced on the basis of the results of direct sequencing. A boundary Tm of 58.0°C allowed us to discriminate HCV genomes into two groups: one with a Tm >58.0°C had no or a low number of nt substitutions, while the other genomes with a Tm <58.0°C had a high number of nt substitutions, corresponding to wild-type in the former and mutant-type in the latter in respect of a clinical setting for IFN therapy. Moreover, this MCA assay provided precise discrimination of Tm between clones, reflecting the degree of the genetic complexity of HCV genomes.
Conclusions: This study indicates that the MCA assay is useful to rapidly and simply screen the mutational status of the ISDR of HCV, as well as in using the ISDR as one of the targets for discriminating the genetic complexity of HCV genomes. The MCA assay could also be applicable as a convenient and useful screen of the genetic heterogeneity of clones relating to HCV quasispecies.
Clin Chem Lab Med 2008;46:966–73.
©2008 by Walter de Gruyter Berlin New York
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Articles in the same Issue
- Editorial
- CCLM: Evolving to meet the needs of today's laboratory professionals and scientists
- Bacterial detection and blood product contamination
- Editorial: Rapid diagnostic tests to detect pathogenic microorganisms
- Reviews
- Rapid methods for diagnosis of bloodstream infections
- Validation criteria for nucleic acid amplification techniques for bacterial infections
- Incidence of bacterial transmission and transfusion reactions by blood components
- Strategies of bacteria screening in cellular blood components
- Methods for the detection of bacterial contamination in blood products
- Bacteria detection by flow cytometry
- Rapid screening by real-time 16S rDNA PCR for bacterial contamination of blood products
- Microbial safety of cell based medicinal products – what can we learn from cellular blood components?
- Genetics and Molecular Diagnostics
- Screening for genetic heterogeneity in the interferon sensitivity determining region of the hepatitis C virus genome by polymerase chain reaction with melting curve analysis
- Thiopurine S-methyltransferase (TPMT) pharmacogenetics: three new mutations and haplotype analysis in the Estonian population
- Genetic testing for adult-type hypolactasia in Italian families
- Improved real-time detection of the H63D and S65C mutations associated with hereditary hemochromatosis using a SimpleProbe assay format
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- Mediterranean diet and plasma concentration of inflammatory markers in old and very old subjects in the ZINCAGE population study
- Plasma interleukin-1β, -6, -8 and tumor necrosis factor-α as highly informative markers of pelvic inflammatory disease
- The effect of hypothyroidism, hyperthyroidism, and their treatment on parameters of oxidative stress and antioxidant status
- Breath isoprene – aspects of normal physiology related to age, gender and cholesterol profile as determined in a proton transfer reaction mass spectrometry study
- Prohormone brain natriuretic peptide (proBNP), BNP and N-terminal-proBNP circulating levels in chronic hemodialysis patients. Correlation with ventricular function, fluid removal and effect of hemodiafiltration
- A comparison of two different assays for determining S-100B in serum and urine
- Diagnostic, clinical and laboratory turnaround times in troponin T testing
- Validation and Outcome Studies
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- Letters to the Editor
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