Exon 4 of the human cytomegalovirus (CMV) major immediate-early gene as a target for CMV real-time PCR.
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ABSTRACT:
SUBMITTER: Leruez-Ville M
PROVIDER: S-EPMC2292956 | biostudies-literature | 2008 Apr
REPOSITORIES: biostudies-literature
Publications
Exon 4 of the human cytomegalovirus (CMV) major immediate-early gene as a target for CMV real-time PCR.
Leruez-Ville Marianne M Ducroux Aurélie A Rouzioux Christine C
Journal of clinical microbiology 20080401 4
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Project description:Here we report how variability in the human cytomegalovirus genome sequence may seriously affect the outcome of its molecular diagnosis. A real-time quantitative PCR assay targeting the major immediate-early gene failed to detect the viral load in some, but not all, clinical samples from hematooncological patients. By amplification and sequencing the DNA across the regions targeted by this assay we found a number of nucleotide substitutions which accounted for decreased primer/probe binding. This decreased the sensitivity of the assay up to 1,000-fold.
| S-EPMC1829136 | biostudies-literature
Project description:A duplex quantitative real-time PCR (qPCR) assay was designed to detect both the polymerase gene (pol) and the glycoprotein gene (gB) of cytomegalovirus (CMV). The detection limit of the qPCR was determined to be 1 to 3 copies/reaction and the linear measure interval was 10(3) to 10(8) copies/ml. The qPCR system was compared to the COBAS Amplicor CMV Monitor test (COBAS) by an analysis of 138 plasma samples. Both systems detected CMV in 71 cases and had negative results for 33 samples. In addition, 34 samples were positive by qPCR and negative by the COBAS assay, but in no case was the COBAS result positive and the qPCR result negative. Thus, qPCR detected 48% more positive cases than the COBAS method. For samples with > or = 10(5) copies/ml by qPCR, a saturation effect was seen in the COBAS assay and quantification required dilution. Copy numbers for pol and gB by qPCR generally agreed. However, the reproducibility of qPCR assays and the need for an international standard are discussed. Discrepant copy numbers for pol and gB by qPCR were found for samples from two patients, and sequence analysis revealed that the corresponding CMV strains were mismatched at four nucleotide positions compared with the gB fragment primer sequences. In conclusion, a duplex qPCR assay in a real-time format facilitates quantitative measurements and minimizes the risk of false-negative results.
| S-EPMC404600 | biostudies-literature
Project description:Cell-mediated immunity plays an essential role in the control of infection with the human cytomegalovirus (HCMV). However, only a few CD8(+)-T-cell epitopes are known, with the majority being contained in the pp65 phosphoprotein, which is believed to dominate the CD8(+)-T-cell response to HCMV. Here, we have readdressed the issue of CD8(+) T cells specific for the 72-kDa major immediate-early protein (IE-1), which is nonstructural but is found very early and throughout the replicative cycle. Using a novel flow-cytometric assay, we were able to identify CD8(+)-T-cell epitopes (by IE-1 peptide-specific induction of cytokine synthesis) and simultaneously measure the frequency of cells directed against them. For this purpose, 81 pentadecamer peptides covering the complete 491-amino-acid sequence of IE-1 were tested on peripheral blood mononuclear cells of anti-HCMV immunoglobulin G-seropositive donors. At least 10 new epitopes were identified, and the fine specificity and presenting HLA molecule of the first of them was determined. The frequencies of CD8(+) T cells directed against IE-1 were similar to those directed against pp65 in donors tested with known pp65-derived peptides. Importantly, additional testing of a corresponding set of peptides covering the complete sequence of pp65 on 10 of these donors identified individuals whose CD8(+) T cells recognized IE-1 but not pp65 and vice versa, clearly illustrating that either protein may be a major target. In summary, our results suggest that IE-1 is far more important as a CD8(+)-T-cell target than current opinion suggests.
| S-EPMC112835 | biostudies-literature
Project description:A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.
| S-EPMC87012 | biostudies-literature
Project description:(1) Background: What is the role of serum CMV PCR in the diagnosis of recent primary infection (PI) in pregnant women when IgG avidity is uninformative? (2) Methods: Retrospective cohort study to compare serum versus whole blood CMV PCR. (a) Qualitative assessment: CMV PCR was performed on 123 serum samples and 74 whole blood samples collected from 132 pregnant women with recent CMV PI. PCR positivity rate was used to calculate sensitivity in serum and whole blood. (b) Quantitative assessment: CMV PCR was performed on 72 paired samples of serum and whole blood collected on the same day from 57 patients. (3) Results: In pregnant women, PCR positivity rate was 89% for serum samples versus 100% in whole blood in the case of very recent PI (<15 days), but only 27% in serum versus 68% in whole blood for PI occurring from 6 weeks to 3 months before. Comparing CMV viral loads between serum and whole blood, we determined the limit of CMV DNA detection in serum as 3 log copies/mL (whole blood equivalent). (4) Conclusions: Serum CMV PCR is reliable in confirming PI in cases when only IgM is detected. It is therefore a valuable tool in introducing valaciclovir treatment as early as possible to prevent mother-to-child CMV transmission.
| S-EPMC9608498 | biostudies-literature
Project description:The human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 p72 and IE2 p86, are activated by a complex enhancer region (base positions -65 to -550) that operates in a cell type- and differentiation-dependent manner. The expression of MIE genes is required for HCMV replication. Previous studies analyzing functions of MIE promoter-enhancer segments suggest that the distal enhancer region variably modifies MIE promoter activity, depending on cell type, stimuli, or state of differentiation. To further understand the mechanism by which the MIE promoter is regulated, we constructed and analyzed several different recombinant HCMVs that lack the distal enhancer region (-300 to -582, -640, or -1108). In human fibroblasts, the HCMVs without the distal enhancer replicate normally at high multiplicity of infection (MOI) but replicate poorly at low MOI in comparison to wild-type virus (WT) or HCMVs that lack the neighboring upstream unique region and modulator (-582 or -640 to -1108). The growth aberrancy was normalized after restoring the distal enhancer in a virus lacking this region. For HCMVs without a distal enhancer, the impairment in replication at low MOI corresponds to a deficiency in production of MIE RNAs compared to WT or virus lacking the unique region and modulator. An underproduction of viral US3 RNA was also evident at low MOI. Whether lower production of IE1 p72 and IE2 p86 causes a reduction in expression of the immediate-early (IE) class US3 gene remains to be determined. We conclude that the MIE distal enhancer region possesses a mechanism for augmenting viral IE gene expression and genome replication at low MOI, but this regulatory function is unnecessary at high MOI.
| S-EPMC111634 | biostudies-literature
Project description:Quantitative real-time PCR (QRT-PCR) has been widely implemented for clinical viral load testing, but a lack of standardization and relatively poor precision have hindered its usefulness. Digital PCR offers highly precise, direct quantification without requiring a calibration curve. Performance characteristics of real-time PCR were compared to those of droplet digital PCR (ddPCR) for cytomegalovirus (CMV) load testing. Tenfold serial dilutions of the World Health Organization (WHO) and the National Institute of Standards and Technology (NIST) CMV quantitative standards were tested, together with the AcroMetrix CMV tc panel (Life Technologies, Carlsbad, CA) and 50 human plasma specimens. Each method was evaluated using all three standards for quantitative linearity, lower limit of detection (LOD), and accuracy. Quantitative correlation, mean viral load, and variability were compared. Real-time PCR showed somewhat higher sensitivity than ddPCR (LODs, 3 log(10) versus 4 log(10) copies/ml and IU/ml for NIST and WHO standards, respectively). Both methods showed a high degree of linearity and quantitative correlation for standards (R(2) ≥ 0.98 in each of 6 regression models) and clinical samples (R(2) = 0.93) across their detectable ranges. For higher concentrations, ddPCR showed less variability than QRT-PCR for the WHO standards and AcroMetrix standards (P < 0.05). QRT-PCR showed less variability and greater sensitivity than did ddPCR in clinical samples. Both digital and real-time PCR provide accurate CMV load data over a wide linear dynamic range. Digital PCR may provide an opportunity to reduce the quantitative variability currently seen using real-time PCR, but methods need to be further optimized to match the sensitivity of real-time PCR.
| S-EPMC3553899 | biostudies-literature
Project description:Human cytomegalovirus (HCMV) is the leading congenital infection agent in the world. The importance of screening this infection has been debated, as 10-15% of the asymptomatic newborns with HCMV at birth will present late sequelae. The aim of this study was to test the feasibility of using saliva pools from newborns in a screening program for congenital HCMV infection, in two Portuguese hospitals. The screening was based on the use of pools of 10 saliva samples for detection of viral DNA by real-time PCR. Whenever there was a positive pool, the samples were tested individually, and for each positive sample the result was confirmed with a urine sample collected in the first 2 weeks of life. The study involved 1492 newborns. One hundred and fifty pools were screened, with 14 positive results in saliva, but only 10 were confirmed in urine samples, giving a prevalence of congenital HCMV infection in both hospitals of 0.67% (CI95% 0.36 to 1.23%).Conclusion: The overall prevalence of congenital HCMV infection in both hospitals was 0.67%. The use of saliva pools proved to be effective for the screening of this congenital infection, allowing timely screening and confirmation in a large population, with associated cost reduction. What is Known: • Newborn screening for HCMV is desirable. • Saliva is a good and practical sample. What is New: • The feasibility of using saliva pools for a large-scale screening. • The cost reduction of this strategy.
| S-EPMC7940150 | biostudies-literature
Project description:We created a multiplex, quantitative, real-time PCR assay that amplifies cytomegalovirus (CMV) and human DNA in the same reaction tube, allowing for a viral load determination that is normalized to measured human DNA. The assay targets a conserved region of the CMV DNA polymerase gene that is not affected by known drug resistance mutations. All 36 strains of CMV detected by culture or qualitative PCR in a population of lung transplant recipients were detected. The assay detected 1 to 10 copies of CMV plasmid DNA. The analytic sensitivity was not affected by the presence of DNA from 10(6) human cells but was reduced approximately 10-fold by alkaline lysates of leukocyte preparations. CMV quantitation was linear over a range of 10(1) to 10(6) copies. The intraassay and interassay coefficients of variation were 29 and 40%. Human DNA was regularly detected in patient plasma samples, and the amount was increased by storage of blood at room temperature before plasma separation and by plasma separation techniques that allowed leukocyte contamination. Applied to whole blood, the assay provides a measurement of CMV DNA in relation to cellular content without a need for cell counting procedures. Applied to plasma, the assay can reveal artifactual increases in plasma CMV levels resulting from leukocyte contamination. Further study of the utility of this assay to monitor patient populations at risk for CMV disease is warranted.
| S-EPMC120584 | biostudies-literature
Project description:Interventions: Patients with the detection of either cytomegalic cells or IHC-CMV cells were diagnosed with CMV colitis.,Patients older than 18 years with clinical suspicion of CMV colitis but negative biopsy for CMV.,Asymptomatic patients underwent colonoscopy for screening.;Diagnostic,Diagnostic,Diagnostic;CMV colitis,Non-CMV colitis ,Healthy volunteers
Primary outcome(s): Diagnostic performance After colonoscopy Sensitivity, Specificity
| 2756739 | ecrin-mdr-crc