title: Development of an In-House TaqMan Real Time RT-PCR Assay to Quantify Hepatitis C Virus RNA in Serum and Peripheral Blood Mononuclear Cells in Patients With Chronic Hepatitis C Virus Infection.
abstract: BACKGROUND
  Viral load measurements are commonly used to monitor HCV infection in patients with chronic diseases or determining the number of HCV-genomes 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.
  
  
  OBJECTIVE
  The aim of this study was to develop a highly specific, sensitive, and reproducible in-house quantitative PCR using specific primers and probe cited in highly conservative region of HCV genome that allows simultaneous detection of HCV genotypes 1 - 4.
  
  
  METHODS
  In this study, three sets of primer pairs and a TaqMan probe for amplification and detection of selected region within 5'-non-coding (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. Real-time 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.
  
  
  RESULTS
  The lower limit detection of this in-house HCV real-time RT-PCR was determined as 100 RNA copies/mL. Inter- and intra-assay coefficient of variation (CV) of this in-house HCV real-time RT-PCR 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 × 10(6) ± 0.31 to 2.7 × 10(5) ± 0.46 copies/mL in serum samples and 5 × 10(2) ± 0.36 to 4.0 × 10(3) ± 0.51 copies/10(6) cells/mL of PBMCs.
  
  
  CONCLUSIONS
  The quite sensitive in-house TaqMan real time RT-PCR assay was able to detect and quantify all four main HCV genotypes prevailing around all geographical regions of Iran.
authors.affiliation: Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Bahman
authors.initials: B
authors.lastname: Khalvati Fahlyani
authors.affiliation: Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran ; Department of Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Abbas
authors.initials: A
authors.lastname: Behzad-Behbahani
authors.affiliation: Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Seiied Alireza
authors.initials: SA
authors.lastname: Taghavi
authors.affiliation: Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran ; Department of Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Farhadi
authors.affiliation: Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran ; Department of Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Saeede
authors.initials: S
authors.lastname: Salehi
authors.affiliation: Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran ; Department of Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Setare
authors.initials: S
authors.lastname: Adibzadeh
authors.affiliation: Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Farzaneh
authors.initials: F
authors.lastname: Aboualizadeh
authors.affiliation: Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Parniyan
authors.initials: P
authors.lastname: Alavi
authors.affiliation: Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Negin
authors.initials: N
authors.lastname: Nikouyan
authors.affiliation: Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Okhovat
authors.affiliation: Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Reza
authors.initials: R
authors.lastname: Ranjbaran
authors.affiliation: Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Gholam Reza
authors.initials: GR
authors.lastname: Rafiei Dehbidi
authors.affiliation: Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Arash
authors.initials: A
authors.lastname: Shakibzadeh
keywords: Hepatitis C Virus; Mononuclear Leukocytes; Real-Time Polymerase Chain Reaction