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.Objectives: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.Materials and 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 × 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.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.

Screening haemodialysis patients for hepatitis C in Vietnam: The inconsistency between common hepatitis C virus serological and virological tests

“…An in‐house quantitative HCV PCR was developed for research purpose and validated with the lower limit of detection was 93 IU/mL of HCV‐RNA. The quality of all these assays is described in detail elsewhere . All PCR positive results were tested for HCV genotype by TRUGENE HCV 5′NC Genotyping kit (SIEMENS) that had a lower limit of detection of 5000 IU/mL of HCV‐RNA .…”

“…The same qualified laboratory technician who was blinded to sample source tested all samples at an ISO 15189:2012 accredited medical and research laboratory . Because the minimum window period for a positive anti‐HCV test is 1‐1.5 months post‐infection we used a two‐monthly screening interval to test anti‐HCV against tests including HCV‐coreAg, HCV‐RNA and HCV genotyping tests that are recognised as effective in identifying HCV early …”

“…The detection limit of HCV‐coreAg assay corresponded to 600‐1000 IU/mL of HCV‐RNA . An in‐house quantitative HCV PCR was developed for research purpose and validated with the lower limit of detection was 93 IU/mL of HCV‐RNA. The quality of all these assays is described in detail elsewhere .…”

Antimicrobial Efficacy of a Novel Antibiotic-Eluting Injectable Platelet-Rich Fibrin Scaffold against a Dual-Species Biofilm in an Infected Immature Root Canal Model

“…Real-time PCR technology provides the possibility of absolute or relative sample quantification analysis [ 32 ]. Absolute quantification determines the amount of a target sequence by back-calculation from the standard curve.…”

An application of competitive reporter monitored amplification (CMA) for rapid detection of single nucleotide polymorphisms (SNPs)

“…The suitability of SNP genotyping methods for the clinical diagnosis is also depended on their sensitivity and accuracy [ 21 ]. The detection of MTBC DNA was possible with a sensitivity down to 50 copies per reaction with the described molecular assay which is comparable to standard SNP detection methods with molecular beacons [ 22 , 23 ] and TaqMan probes [ 24 , 25 ]. With the CMA-based SNP assay all genotypes of the sequenced isolates could be correctly identified ( Fig 3 and S7 Table ) indicating that the test accuracy is as high as for alternative SNP methods.…”