A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping
Rev. Soc. Bras. Med. Trop
;
47(3): 287-294, May-Jun/2014. tab, graf
Artículo
en Inglés
| LILACS
| ID: lil-716399
ABSTRACT
Introduction Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5′ untranslated region (5′UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Methods Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. Results The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. Conclusions A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping. .
Texto completo:
Disponible
Índice:
LILACS (Américas)
Asunto principal:
ARN Viral
/
Hepatitis C
/
Hepacivirus
Tipo de estudio:
Estudio diagnóstico
Límite:
Humanos
Idioma:
Inglés
Revista:
Rev. Soc. Bras. Med. Trop
Asunto de la revista:
Medicina Tropical
Año:
2014
Tipo del documento:
Artículo
País de afiliación:
Brasil
Institución/País de afiliación:
Universidade Luterana do Brasil/BR
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