An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

Abstract

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.

A New Method for Next-Generation Sequencing of the Full Hepatitis B Virus Genome from A Clinical Specimen: Impact for Virus Genotyping

“…The DNA concentration was analyzed using a fluorometer (Qubit 2.0, Invitrogen, Carlsbad, CA, USA) with a DNA High Sensitivity Assay kit (Invitrogen, Carlsbad, CA, USA). For all clinical samples, DNA was used as the template for qPCR reactions to verify whether DNAse treatment can affect viral DNA amplification, according to an in-house real- time PCR assay with some modifications [ 19 ]. The qPCR reaction using GoTaq ® qPCR Master Mix (Promega, A6001) was performed on an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster city, CA, USA) in a 20 μL reaction solution containing 10μL master mix, 0.7 μL of each forward primer (F1primer 5′-CAACCTCTTGTCCTCCAACTTGT-3′; F2 primer 5′-AACCTCCTGTCCTCCAACTTGT-3′ and F3 primer 5′-CAACCTGTTGTCCTCCAATTTGT-3′), 2 μL of reverse primer (R primer 5′-GATGAGGCATAGCAGCAGGAT-3′), and 5 μL of DNA.…”

Novel evidence indicates the presence and replication of hepatitis B virus in breast cancer tissue

“…Data were quantified using its own Optiquant software. HBV DNA was also quantified by performing quantitative PCR using a TaqMan probe (Probe-HBV) and primers (Primer-HBV-S-F and Primer-HBV-S-R; Table II) (19). To perform PCR according to the manufacturer's protocol, HBV DNA was extracted from 600 µl of serum or cell-cultured supernatant using a DNA Extraction Reagent of HBV Fluorescence Quantitative Polymerase Chain Reaction kit (Fosun Pharma Diagnostics, China).…”

Immunovirological Assessment of HIV-Infected Patients and Phylogenetic Analysis of the Virus in Northeast of Iran

“…The quantification of HIV RNA in plasma has been introduced for determining the disease progression and the beginning of treatment time (15,16). Besides, a diversity of sophisticated molecular techniques, such as TaqMan Real-Time PCR (qRT-PCR) and COBAS Amplicor monitor test, are applied for the quantitation of the viral load (17)(18)(19). The clinical variability and phenotypic heterogeneity in clinical manifestations of HIV-related symptoms and the duration of latency in a given population are surprisingly high (20).…”