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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

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Development of a low-cost real-time reverse transcriptase-polymerase chain reaction technique for the detection and quantification of hepatitis C viral load

Kiana Shahzamani1 / Shahin Merat1 / Houri Rezvan2 / Siamak Mirab-Samiee3 / Hooman Khademi1 / Reza Malekzadeh1 / Farzaneh Sabahi4

1Digestive Disease Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran

2Iranian Blood Transfusion Organization/Research Center, Tehran, Iran

3Food and Control Research Laboratories, Tehran, Iran

4Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Corresponding author: Farzaneh Sabahi, PhD, Department of Virology, Faculty of Medical Sciences, P.O. Box 14115-331, Tehran, Iran Phone: +98 21 8288 3880, Fax: +98 21 8288 4555,

Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 48, Issue 6, Pages 777–784, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2010.134, March 2010

Publication History

Received:
2009-11-21
Accepted:
2010-01-08
Published Online:
2010-03-11

Abstract

Background: It is necessary to develop a highly specific and sensitive assay to quantify the exact amount of hepatitis C virus (HCV) RNA in blood of patients with hepatitis C. For this reason, a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for quantification of HCV RNA in human plasma was developed.

Methods: A pair of primers as well as hybridization probes were selected. A real-time RT-PCR was set up and optimized. To establish the sensitivity of the assay, a serial dilution of HCV standards and reference sera, including the six major HCV genotypes, was used. The performance of the assay was evaluated with 191 known HCV-RNA positive and 100 negative samples.

Results: The real-time assay had a sensitivity of 50 IU/mL, with a dynamic range of detection between 103 and 106 IU/mL. The coefficients of variation of threshold cycle values in intra- and inter-day-runs were <1.77% and 3.40%, respectively. Measurement of HCV-RNA positive samples yielded reproducible data with 100% specificity.

Conclusions: The high sensitivity, simplicity, reproducibility, wide dynamic range, and low cost of this real-time HCV RNA quantification makes this method especially suitable for monitoring viral load during therapy and tailoring of treatment schedules accordingly.

Clin Chem Lab Med 2010;48:777–84.

Keywords: hepatitis C virus; hybridization probe; real-time reverse transcriptase-PCR; viral load

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