Development of an InHouse TaqMan Real Time RTPCR Assay to Quantify Hepatitis C Virus RNA in Serum and Peripheral Blood Mononuclear Cells in Patients With Chronic Hepatitis C Virus Infection

Viral load measurements are commonly used to monitor HCV infection in patients with chronic diseases or determining the number of HCVgenomes in serum samples of patients after sustained virological response. However, in some patients, HCV viral load in serum samples is too low to be detected by PCR, especially after treatment. The aim of this study was to develop a highly specific, sensitive, and reproducible inhouse quantitative PCR using specific primers and probe cited in highly conservative region of HCV genome that allows simultaneous detection of HCV genotypes 1 4. In this study, three sets of primer pairs and a TaqMan probe for amplification and detection of selected region within 5’noncoding (5’NCR) of four HCV genotypes were used. Using plasmid containing 5’NCR region of HCV, standard curve, threshold, and threshold cycle (CT) values were determined. Realtime and nested PCR were performed on HCV genotypes 1 4 extracted from plasma and peripheral blood mononuclear cells (PBMCs) samples collected from patients with chronic HCV infection. The lower limit detection of this inhouse HCV realtime RTPCR was determined as 100 RNA copies/mL. Inter and intraassay coefficient of variation (CV) of this inhouse HCV realtime RTPCR ranged from 0.9% to 1.8% and 1.76% to 3.94%, respectively. The viral load of the genotyped samples ranged from 2.0 × 106 ± 0.31 to 2.7 × 105 ± 0.46 copies/mL in serum samples and 5 × 102 ± 0.36 to 4.0 × 103 ± 0.51 copies/106 cells/mL of PBMCs. The quite sensitive inhouse TaqMan real time RTPCR assay was able to detect and quantify all four main HCV genotypes prevailing around all geographical regions of Iran.