Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5′ Noncoding and Nonstructural 5b Genomic Regions
| Autor: | Ramon Planas, Elisa Martró, Lurdes Matas, Gema Fernández, Verónica Saludes, Andrew J. Buckton, Victoria González, Vicenç Ausina |
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| Rok vydání: | 2008 |
| Předmět: |
Serum
Microbiology (medical) Statistics as Topic Genomics Hepacivirus Biology Polymerase Chain Reaction Sensitivity and Specificity chemistry.chemical_compound Viral Envelope Proteins Virology Genotype Humans Typing NS5B Genotyping Genetics Nucleic Acid Hybridization Sequence Analysis DNA Amplicon Hepatitis C Subtyping chemistry RNA Viral Pyrosequencing 5' Untranslated Regions |
| Zdroj: | Journal of Clinical Microbiology. 46:192-197 |
| ISSN: | 1098-660X 0095-1137 |
| Popis: | We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design is such that genotype 1 isolates are typed by amplification targeting the nonstructural 5b (NS5b) subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5′ noncoding region (5′NC) (method 1; HCV genotyping analyte-specific reagent assay). This method was compared with 5′NC reverse hybridization (method 2; InnoLiPA HCV II) and 5′NC sequencing (method 3; Trugene HCV 5′NC). Two hundred ninety-five sera were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic analysis of an NS5b fragment were used to resolve discrepant results. Suspected multiple-genotype infections were confirmed by PCR cloning and pyrosequencing. Even though a 2% rate of indeterminates was obtained with method 1, concordance at the genotype level with results with methods 2 and 3 was high. Among eight discordant results, five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2, and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct) and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45%, and 92% by methods 1, 2, and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within genotype 2 and that most subtype calls were not correct. Although only sequencing-based methods provide the possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret, thus providing a good single-step alternative to more-time-consuming assays. |
| Databáze: | OpenAIRE |
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