Journal Article

Absolute Quantitation of Different Genotypes of Hepatitis C Virus RNA in Clinical Samples by a Modified Real-Time PCR Method

Karina del Carmen Trujillo-Murillo, Diana Cristina Pérez-Ibave, Clara Patricia Ríos-Ibarra, Eda Guadalupe Ramirez-Valles, Ana Rosa Rincón-Sánchez and Ana María Rivas-Estilla

in Laboratory Medicine

Published on behalf of American Society for Clinical Pathology

Volume 42, issue 6, pages 333-337
Published in print June 2011 | ISSN: 0007-5027
Published online October 2015 | e-ISSN: 1943-7730 | DOI: https://dx.doi.org/10.1309/LMHXO54SA8GYPIRH
Absolute Quantitation of Different Genotypes of Hepatitis C Virus RNA in Clinical Samples by a Modified Real-Time PCR Method

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  • Clinical Cytogenetics and Molecular Genetics
  • Molecular and Cell Biology
  • Molecular Biology and Genetics

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Background:

Our aim was to develop a quantitative real-time polymerase chain reaction (qPCR) assay for the quantitation of hepatitis C virus (HCV) RNA samples from patients infected with different HCV genotypes.

Methods:

A standard curve was generated by amplification of serial dilutions of HCV-plasmid (pFKI389-NS3-3′) harboring genotype 1b HCV-subgenomic replicon. Samples from 15 HCV-infected patients (genotypes 1, 2, and 3) were analyzed to quantify HCV-RNA by qPCR with primers and probes specific for the 5′-UTR viral region.

Results:

The HCV qPCR assay had a sensitivity of 100 copies/reaction with a dynamic range of detection between 102–20 × 106 HCV copies. The assay was highly reproducible with a low coefficient of variation. We observed that the HCV genotypes included could be identified by our method.

Conclusions:

Our results showed that this modified qPCR assay provides a valid platform for quantifying HCV-RNA, combining good analytical sensitivity with a wide dynamic range and high reproducibility.

Keywords: hepatitis C virus; HCV-RNA; polymerase chain reaction; qPCR; HCV genotype; untranslated region; nucleotides; threshold cycle

Journal Article.  2425 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics ; Molecular and Cell Biology ; Molecular Biology and Genetics

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