Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PCR assay monitoring”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources
PubMed1992

PreS antigen expression and anti-preS response in hepatitis B virus infections: relationship to serum HBV-DNA, intrahepatic HBcAg, liver damage and specific T-cell response.

The diagnostic value of preS antigens and anti-preS antibodies during Hepatitis B virus (HBV) infections have not yet been clearly elucidated. Therefore, the objectives of this study were: 1) to better understand the clinical significance of the expression of both preS1 and preS2 antigens (preS1Ag and preS2Ag) in the serum of chronic HBsAg carriers, and 2) to define the respective role of antibody responses to HBs-, preS2- and preS1-specific determinants in the course of acute hepatitis B (AH-B) infections with different outcomes. Our data showed that the serum preS1Ag/HBsAg ratio correlated well with the level of viral replication (serum HBV-DNA as monitored by PCR assay and liver HBcAg), especially in anti-HBe positive chronic carriers. The complete eradication of virions required a persistent antibody response to conformation-dependent HBs-epitopes, temporally associated with a vigorous T cell response to nucleocapsid antigens. Recovery from hepatitis B can be achieved when there is no early antibody response to preS2- and preS1-proteins, which was found to be transient, concomitant with a flare-up of the liver disease, and preceding anti-HBs production. Information on the patterns of preS antigens and their antibodies remained clouded because of the varying specificities and sensitivities of research methods used in studies to date. We have, therefore, developed original Polyclonal-Monoclonal RadioImmunoAssays (PAb-MAb RIAs) by using monoclonal antibodies (MAbs) having previously well-defined specificities. We could thus detect and quantify simultaneously the three distinct antigenicities of the HBV envelope, HBsAg, preS2Ag and preS1Ag, with the same sensitivity. In this way, the preS1Ag/HBsAg and preS2Ag/HBsAg ratios can be calculated to estimate the serum expression of both preS1Ag and preS2Ag in relation to total HBsAg activity during different stages of chronic HBV infection. For optimal management of the state of HBV replication in chronic viral infection, the detection of HBV-DNA in serum was monitored by the Polymerase Chain Reaction (PCR) assay. We extended our work by investigating the clinical significance of antibody response to preS-specific determinants in patients with AH-B showing different outcomes in both natural course or response to alpha-interferon therapy. In a first attempt, we chose to use the Western Immuno-Blotting Assay (WIBA) to obtain a qualitative assessment of the nature of preS antibody responses. Finally, the cell-mediated immune response to HBV antigens was also studied in several patients with self-limited AH-B leading to a relevant finding which may help to clarify the mechanisms responsible for complete clearance of HBV.

Acute Disease↗
PubMed2006

Evaluation of the COBAS Amplicor HCMV Monitor for early detection and monitoring of human cytomegalovirus infection after allogeneic stem cell transplantation.

Early diagnosis of human cytomegalovirus (HCMV) infection and the introduction of preemptive antiviral therapy have reduced HCMV-related mortality after allogeneic stem cell transplantation. A critical goal remains stratifying risk profiles and minimizing potential harm owing to antiviral overtreatment. We compared the commercially available standardized COBAS Amplicor CMV Monitor (CACM) to an in-house PCR assay, for the monitoring of HCMV infection. Seventy-two patients were surveyed by an in-house PCR of whole blood, quantitative viral load assessment by CACM and virus culture assays in a prospective and a retrospective study. A high concordance between CACM and PCR was documented. The viral load at onset correlated with the peak viral load (Spearman rank correlation R=0.634, P=0.0004). In patients developing HCMV disease, both viral loads were in trend higher (P=0.823, respectively P=0.053), and the viremic episodes longer (P=0.015), as compared to asymptomatically HCMV-infected patients. The serological pre-transplant status was the major risk factor for the development of HCMV disease, showing highest risk for seropositive patients receiving a seronegative graft, whereas donor type (related or unrelated) and graft type (bone marrow or peripheral blood mobilized stem cells) did not have an influence. HCMV infection proved to be a risk factor for the development of non-viral opportunistic infections (P=0.002).

Adult↗
PubMed2002

A sensitive, quantitative assay for human immunodeficiency virus type 1 integration.

Quantitative methods to measure human immunodeficiency virus type 1 (HIV-1) integration promise to be important tools in dissecting the mechanisms whereby latent reservoirs of provirus are established, most notably in the resting T cells of patients receiving antiretroviral therapy. Here we describe a fluorescence-monitored, nested PCR assay that is able to quantify the relatively rare integration events that occur within these cells. Following DNA extraction, a nonkinetic preamplification step is performed with primers that bind genomic Alu elements and HIV-1 gag sequences, under conditions where primers, deoxynucleoside triphosphates, and enzyme are not limiting. This is followed by a kinetic PCR that quantitates HIV-1 long terminal repeat sequences. A T-cell-based integration standard which reflects the randomness of HIV-1 integration is also described. The assay is 10 to 100 times more sensitive than previously reported quantitative Alu PCR-based integration assays. It is specific for integration events, since no proviruses are detected in cells infected either in the presence of an integrase inhibitor or with an integrase-deficient virus. This method promises to provide important new insights into the processes underlying the accumulation and persistence of latent HIV-1 reservoirs and may eventually be useful clinically in monitoring the eradication of latent virus by novel therapies.

CD4-Positive T-Lymphocytes↗
PubMed2004

A high-throughput method to monitor the expression of microRNA precursors.

microRNAs (miRNAs) are small, functional, non-coding RNAs. miRNAs are transcribed as long primary transcripts (primary precursors) that are processed to the approximately 75 nt precursors (pre-miRNAs) by the nuclear enzyme Drosha. The approximately 22 nt mature miRNA is processed from the pre-miRNA by the RNase III Dicer. The vast majority of published studies to date have used northern blotting to detect the expression of miRNAs. We describe here a sensitive, high throughput, real-time PCR assay to monitor the expression of miRNA precursors. Gene-specific primers and reverse transcriptase were used to convert the primary precursors and pre-miRNAs to cDNA. The expression of 23 miRNA precursors in six human cancer cell lines was assayed using the PCR assay. The miRNA precursors accumulated to different levels when compared with each other or when a single precursor is compared in the various cell lines. The precursor expression profile of three miRNAs determined by the PCR assay was identical to the mature miRNA expression profile determined by northern blotting. We propose that the PCR assay may be scaled up to include all of the 150+ known human miRNA genes and can easily be adaptable to other organisms such as plants, Caenorhabditis elegans and Drosophila.

Blotting, Northern↗
PubMed2005

Development of a real-time PCR assay using SYBR Green chemistry for monitoring Marek's disease virus genome load in feather tips.

Feather follicles of birds infected with Marek's disease virus (MDV) serve as the sole source of infectious virus particles. The present study was aimed at developing a SYBR Green real-time PCR assay to detect and quantify MDV loads in feather tips targeting meq gene of the virus. The assay had a dynamic range of 8 logs, mean inter- and intra-assay coefficient variation (CV) of <5% and minimum detection limit of 15 MDV genome copies when plasmid DNA was used as the template. The sensitivity of the assay was compared with that of the conventional PCR technique and found to be 2.5-10 times more sensitive than the conventional PCR technique. The assay was validated using feather tip DNA preparations derived from chickens infected with 250 plaque forming units (PFU) of RB1B strain of MDV and sampled on days 7, 14, 21 and 28 post-infection (p.i.) along with uninfected chickens. MDV genome was quantifiable in feather tips of infected birds by day 7 p.i. and the number of MDV copies peaked by day 14 p.i., but then gradually decreased by day 28. This reliable real-time PCR assay may be used for monitoring MDV genome loads in tissues of experimentally or naturally infected birds.

Animals↗
PubMed1996

Evaluation of a novel PCR-based diagnostic assay for detection of Mycobacterium tuberculosis in sputum samples.

We report on a PCR-based assay we have developed for the detection of Mycobacterium tuberculosis in sputum samples. One hundred sputum specimens, which included 34 culture-positive and 66 culture-negative specimens, were evaluated with this system. Of the 34 culture-positive specimens, 31 were PCR positive, and 60 of the culture-negative specimens were PCR negative. An internal standard has been included in the assay system to monitor PCR inhibition and to confirm the reliability of the PCR assay.

Biological Assay↗
PubMed2003

Power analysis for real-time PCR quantification of genes in activated sludge and analysis of the variability introduced by DNA extraction.

The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.

Bacteria↗
PubMed2003

Comparison of LightCycler-based PCR, COBAS amplicor CMV monitor, and pp65 antigenemia assays for quantitative measurement of cytomegalovirus viral load in peripheral blood specimens from patients after solid organ transplantation.

In order to evaluate the LightCycler-based PCR (LC-PCR) as a diagnostic assay technique, a classical pp65 antigenemia assay and the commercially available COBAS Amplicor CMV Monitor (CACM) assay were compared to the LC-PCR assay for the detection and quantitation of cytomegalovirus (CMV) load in 404 parallel specimens of peripheral blood from 66 patients after solid organ transplantation. A good correlation existed among these three assays (r congruent with 0.6, P < 0.0001). The LC-PCR assay was the most sensitive (54% of specimens positive) compared to the CACM (48.6%) and the pp65 antigenemia (26%) assays. The LC-PCR assay detected all samples found positive by using both the CMV pp65 antigenemia assay and the CACM assay. The LC-PCR also had the widest dynamic range (from 250 to 10(7) DNA copies/ml of plasma). No cross-reactions were found among CMV and Epstein-Barr virus, varicella-zoster virus, or herpes simplex virus in the LC-PCR by using amplification with specifically designed primer pairs. Precision, expressed as the coefficient of variation, was <3% with standard DNA from cell cultures and between 6.55 and 14.1% with clinical specimens in repeat LC-PCR runs. One run of the LC-PCR took half of the time required for the semiautomated CACM procedure. Because of its sensitivity, specificity, cost-effectiveness, and simplicity, the LC-PCR assay could replace the pp65 antigenemia and the CACM assays as the preferred technique for the surveillance, diagnosis, and monitoring of response of CMV diseases in high-risk populations.

Cytomegalovirus↗
PubMed2000

Follow-up of chimerism, including T- and B-lymphocytes and granulocytes in children more than one year after allogeneic bone marrow transplantation.

In bone-marrow-transplanted children, early detection of graft failure, relapse, and other potentially treatable problems is facilitated by the use of polymerase chain reaction (PCR) assays that monitor whether blood and marrow cells are of recipient or donor origin. Presence of mixed donor-recipient chimerism (MC) within the first year after BMT frequently correlates with clinical problems. To study if MC detected one year or more post-BMT was also often associated with clinical problems, the chimeric status in 33 children surviving 1-11 yr (median: 2 yr) after BMT was investigated. A PCR with a sensitivity of 1-2%, using fluorescent primers analyzing DNA fragment length polymorphisms, was applied. T- and B-cells and granulocytes were immunomagnetically isolated and tested separately for all patients. Of the 33 patients, of whom 21 had received pretreatment including total body irradiation (TBI), 27 (82%) exhibited full donor chimerism. Six children (18%), four of whom had received pretreatment without TBI, had MC. In three of these children, all with aplastic anemia, isolated T-cell MC had not posed apparent clinical problems. In two patients, both with MC including B-cells, immune hemolytic anemia was observed. A sixth patient with AML presented with MC and relapse. In two of the six children MC was detected only by cell subset analysis. In conclusion, analysis of MC in leukocyte subsets is more informative than analysis of whole blood only and may reveal clinically important variations in the origin of different cell populations. The prevalence of MC is lower after the first year post-BMT, and when present is less often associated with clinical problems.

Adolescent↗
PubMed2005

Development of a method to quantify gene expression levels for glycosylation pathway genes in chinese hamster ovary cell cultures.

Changes in protein glycosylation owing to changes in environmental conditions are not well understood. To better understand these relationships, methods to quantify controlling factors are needed. Because enzymes are translated from genes, the ability to quantify gene expression levels for glycosylation-related enzymes would be advantageous. We developed quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays to monitor gene expression in Chinese hamster ovary (CHO) cells for five terminal glycosylation genes. The five enzymes were sialidase, a putative alpha2,3-sialyltransferase, beta1,4-galactosyltransferase, cytosine monophosphate-sialic acid transporter, and uracil diphosphate-galactosyl transporter. Four of these CHO cell genes were publicly available from GenBank; however, the alpha2,3-sialyltransferase gene for Cricetulus griseus (CHO cell species) was not available and, therefore, was sequenced as a part of this work. The qRT-PCR primers and probes (based on the TaqMan chemistry) were designed and validated for these five genes. The gene expression profiles were obtained for CHO cells producing the recombinant interleukin-4/13 cytokine trap molecule in batch reactors.

Animals↗
PubMed1995

Detection of cytokine transcriptional profiles from bovine peripheral blood mononuclear cells and CD4+ lymphocytes by reverse transcriptase polymerase chain reaction.

Cytokine transcriptional profiles constitute important information about T cell mediated immunity against pathogens. We have developed a reverse transcriptase polymerase chain reaction (RT-PCR) assay to monitor gene expression of bovine T lymphocyte cytokines. Bovine cDNA was reverse transcribed from total RNA and subsequently amplified using primers designed from bovine or murine and human consensus sequences. Cytokine transcription of beta-actin, interleukins (IL) IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)alpha, TNF beta and interferon-gamma was detected from concanavalin A activated peripheral blood mononuclear cells and CD4+ purified T lymphocytes. The assay allows detection of many cytokine mRNA in a species where research has been hindered by lack of commercially available reagents and sequence information. RT-PCR analysis of lymphocyte cytokines will be invaluable for understanding the progression or resolution of disease as a consequence of lymphocyte response to specific antigens.

Animals↗
PubMed2000

Quantitative analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay and the COBAS AMPLICOR CMV MONITOR PCR test after blood and marrow allogeneic transplantation.

The performance of a commercially available qualitative PCR test for plasma (AMPLICOR CMV Test; Roche Diagnostics) and a quantitative PCR test for plasma and leukocytes (COBAS AMPLICOR CMV MONITOR Test; Roche Diagnostics) was evaluated with samples from 50 blood or marrow allogeneic transplant recipients who received short courses of sequential ganciclovir therapy (2 weeks intravenously followed by 2 weeks orally) based on a positive cytomegalovirus (CMV) pp65 antigenemia (AG) assay. The number of persons with a positive CMV test was significantly higher for leukocyte-based assays (AG, 67.5%; PCR, 62.5%) compared to both quantitative and qualitative PCR tests of plasma (42.5 and 35%, respectively). One person developed CMV disease during the study despite a negative AG assay; in this particular case, all PCR assays were found to be positive 10 days before his death. There was a trend for earlier positivity after transplantation and more rapid negativity after initiation of ganciclovir for the tests performed on leukocytes. The mean number of CMV copies as assessed by PCR was significantly higher in leukocytes than in plasma (P = 0.02). Overall, excellent agreement (kappa coefficient, >0.75) was found only between the two PCR assays (qualitative and quantitative) based on plasma. These results suggest that either the pp65 AG assay or the COBAS AMPLICOR CMV MONITOR Test using leukocytes could be used to safely monitor CMV viremia in related allogeneic blood or marrow transplant recipients. Such a strategy will result in preemptive treatment for about two-thirds of the persons with a relatively low rate (<33%) of secondary viremic episodes following short courses of ganciclovir therapy.

Bone Marrow Transplantation↗
PubMed1999

Quantification of hepatitis C virus by TaqMan PCR: comparison with HCV Amplicor Monitor assay.

The quantitation of serum levels of hepatitis C virus RNA in chronic hepatitis C has been regarded as one of the most important indicators for the outcome of interferon therapy. A new method was used for quantitating the copy number of hepatitis C virus RNA using TaqMan polymerase chain reaction and for comparing the ability and usefulness of this assay with Amplicor Monitor assay in 138 patients. The detection range of hepatitis C virus RNA by TaqMan polymerase chain reaction was from 2 x 10(3) to 2 x 10(8) copies/ml. Hepatitis C virus RNA was detectable in 128 cases (92.8%) and undetectable in 10 cases (7.2%) by this method. The RNA levels measured by Amplicor Monitor assay correlated significantly with those measured by TaqMan polymerase chain reaction assay and the sensitivity of the two assays was almost equal. Thus, TaqMan polymerase chain reaction assay appears sufficiently sensitive for the evaluation of hepatitis C virus RNA and would be useful for the diagnosis and management of hepatitis C virus infection.

Adolescent↗
PubMed1995

RNA editing of the coI mRNA throughout the life cycle of Physarum polycephalum.

Editing of RNA via the insertion, deletion or substitution of genetic information affects gene expression in a variety of systems. Previous characterization of the Physarum polycephalum cytochrome c oxidase subunit I (coI) mRNA revealed that both nucleotide insertions and base substitutions occur during the maturation of this mitochondrial message. Both types of editing are known to be developmentally regulated in other systems, including mammals and trypanosomatids. Here we show that the coI mRNA present in Physarum mitochondria is edited via specific nucleotide insertions and C to U conversions at every stage of the life cycle. Primer extension sequencing of the RNA indicates that this editing is both accurate and efficient. Using a sensitive RT-PCR assay to monitor the extent of editing at individual sites of C insertion, we estimate that greater than 98% of the steady-state amount of coI mRNA is edited throughout the Physarum developmental cycle.

Animals↗
PubMed2003

DNA replicative functions of highly-expressed, codon-optimized human papillomavirus proteins E1 and E2.

Human papillomavirus (HPV) DNA replication requires functional HPV early (E) proteins E1 and E2. To determine the biological activity of HPV 16 E1 and E2 mutant proteins under consideration as vaccine candidates, we developed a sensitive real time PCR assay that monitors HPV origin-of-replication-driven DNA synthesis. The assay was used to determine the DNA replicative functions of highly-expressed, codon-optimized HPV 16 E1 and E2 wild type and mutant proteins in transient transfections. Under the assay conditions, the HPV 16 E1 mutant (W439R, G482D) did not support HPV origin-driven DNA synthesis. In contrast, however, an HPV 16 E2 mutant bearing an E39A substitution, reported previously to be severely compromised for DNA replication, was found to be reduced only two-fold in activity and, therefore, considered not sufficiently inactivated for use in vaccines that depend on endogenous protein expression.

Amino Acid Substitution↗
PubMed2000

The number of HIV DNA-infected mononuclear cells is reduced under HAART plus recombinant IL-2. IRHAN Study Group.

It is common opinion that, in addition to potent antiretroviral regimens which effectively reduce plasma viremia, new strategies should be developed to ensure the reduction of cell-associated HIV DNA load together with HIV RNA plasma levels. The present study explored whether the number of provirus-infected cells can be reduced by combined antiviral and immunomodulatory regimens. Thus, 14 naive patients (with CD4 > 400/microl and plasma HIV RNA copies > 5000/ml) were randomly assigned to receive highly active antiretroviral therapy (HAART) alone or HAART plus rIL-2. Plasma viremia (measured by a commercial RT-PCR assay) and the number of provirus-infected cells (measured by an endpoint cell dilution PCR assay) were monitored at the enrollment and after 12 weeks of treatment. The results indicate that while HAART and HAART plus rIL-2 are both able to significantly reduce plasma viremia after 12 weeks of treatment, a significant reduction of the number of provirus-infected cells can be achieved only by treatment with HAART plus rIL-2.

Anti-HIV Agents↗
PubMed1997

Quantification of CMV viraemia in a case of transfusion-related graft-versus-host disease associated with purine analogue treatment.

We report a patient who developed transfusion-associated graft-versus-host-disease (GvHD) and concurrent cytomegalovirus (CMV) infection, both complications thought to be related to severe T lymphocyte depletion induced by treatment with a purine analogue drug, fludarabine. CMV viraemia was detected by qualitative PCR and the viral load in positive samples was measured using a fully quantitative PCR assay. This quantitative assay enabled the evaluation of the efficacy of antiviral interventions based on the qualitative PCR result. The case illustrates the risks associated with the use of purine analogue drugs, as well as the value of quantitative CMV PCR assays for monitoring CMV infection in immunocompromised patients.

Acute Disease↗
PubMed2006

Only a few fungal species dominate highly diverse mycofloras associated with the common reed.

Plants are naturally colonized by many fungal species that produce effects ranging from beneficial to pathogenic. However, how many of these fungi are linked with a single host plant has not been determined. Furthermore, the composition of plant-associated fungal communities has not been rigorously determined. We investigated these essential issues by employing the perennial wetland reed Phragmites australis as a model. DNA extracted from roots, rhizomes, stems, and leaves was used for amplification and cloning of internal transcribed spacer rRNA gene fragments originating from reed-associated fungi. A total of 1,991 clones from 15 clone libraries were differentiated by restriction fragment length polymorphism analyses into 345 operational taxonomical units (OTUs). Nonparametric estimators for total richness (Chao1 and ACE) and also a parametric log normal model predicted a total of about 750 OTUs if the libraries were infinite. Sixty-two percent of the OTUs sequenced were novel at a threshold of 3%. Several of these OTUs represented undocumented fungal species, which also included higher taxonomic levels. In spite of the high diversity of the OTUs, the mycofloras of vegetative organs were dominated by just a few typical fungi, which suggested that competition and niche differentiation influence the composition of plant-associated fungal communities. This suggestion was independently supported by the results of nested PCR assays specifically monitoring two OTUs over 3 years, which revealed significant preferences for host habitat and host organ.

Base Sequence↗