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

Research paper by Kiana K Shahzamani, Shahin S Merat, Houri H Rezvan, Siamak S Mirab-Samiee, Hooman H Khademi, Reza R Malekzadeh, Farzaneh F Sabahi

Indexed on: 12 Mar '10Published on: 12 Mar '10Published in: Clinical chemistry and laboratory medicine


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.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.The real-time assay had a sensitivity of 50 IU/mL, with a dynamic range of detection between 10(3) and 10(6) 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.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.