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Results for “Quantitative PCR”

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PubMed1999

The development of a quantitative PCR ELISA to determine HCMV DNAaemia levels in heart, heart/lung and lung transplant recipients.

The adaptation of the polymerase chain reaction (PCR) enzyme-linked immunosorbent assay (ELISA) using a co-amplified DNA standard to quantitate the human cytomegalovirus (HCMV) glycoprotein B (gB) gene in clinical samples is described. The PCR ELISA is a solution hybridisation system with colorimetric end stage detection of amplicons. A DNA internal standard (IS) was designed by replacing a probe sequence used currently within the gB region with a heterogeneous sequence, allowing co-amplification with the same oligonucleotide primer sets and differentiation by probe hybridisation. Two DNA fragments homologous to the gB and IS sequences were generated and used for co-amplification studies to construct a standard curve. From this the copy number of the gB gene present in clinical samples could be interpolated. Co-amplification with 1000 IS copies allowed quantitation of 10-1000000 gB DNA copies in a single PCR. This assay was validated subsequently using blood samples tested by the HCMV antigenaemia assay and showed a trend of increasing HCMV DNAaemia with increasing antigenaemia levels. This rapid assay avoids using gel electrophoresis and cumbersome quantitative systems. It has the potential for early identification of patients at high risk of developing HCMV disease, and for therapeutic monitoring.

Base Sequence↗
PubMed2006

Application of a methylation gene panel by quantitative PCR for lung cancers.

Detection of lung cancer at early stages could potentially increase survival rates. One promising approach is the application of suitable lung cancer-specific biomarkers to specimens obtained by non-invasive methods. Thus far, clinically useful biomarkers that have high sensitivity have proven elusive. Certain genes, which are involved in cellular pathways such as signal transduction, apoptosis, cell to cell communication, cell cycles and cytokine signaling are down-regulated in cancers and may be considered as potential tumor suppressor genes. Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor genes in many kinds of human cancers. Using quantitative real time PCR, we tested 11 genes (3-OST-2, RASSF1A, DcR1, DcR2, P16, DAPK, APC, ECAD, HCAD, SOCS1, SOCS3) for levels of methylation within their promoter sequences in non-small cell lung cancers (NSCLC), adjacent non-malignant lung tissues, in peripheral blood mononuclear cells (PBMC) from cancer free patients, in sputum of cancer patients and controls. Of all the 11 genes tested 3-OST-2 showed the highest levels of promoter methylation in tumors combined with lowest levels of promoter methylation in control tissues. 3-OST-2 followed by, RASSF1A showed increased levels of methylation with advanced tumor stage (P<0.05). Thus, quantitative analysis of 3-OST-2 and RASSF1A methylation appears to be a promising biomarker assay for NSCLC and should be further explored in a clinical study. Our preliminary data on the analysis of sputum DNA specimens from cancer patients further support these observations.

Adenocarcinoma↗
PubMed1997

Rapid detection of trisomy 21 by homologous gene quantitative PCR (HGQ-PCR).

Down's syndrome results from the production of three copies of chromosome 21 within a cell. We have devised a method termed the homologous gene quantitative polymerase chain reaction (HGQ-PCR), which uses one pair of primers and which can directly identify the additional copy of chromosome 21 by simultaneously amplifying two highly homologous genes of the human liver-type phosphofructokinase located on chromosome 21 (PFKL-CH21) and the human muscle-type phosphofructokinase located on chromosome 1 (PFKM-CH1) for self-detecting determination. On analysis of 34 cases of Down's syndrome, including two cases of unbalanced translocation 46, XY, der (14; 21) (q10; q10), + 21, and 100 normal individuals, the relative ratio of the PFKM-CH1/PFKL-CH21 product was 1.33 +/- 0.323 (mean +/- SD) and 0.40 +/- 0.16 (mean +/- SD) for disomy DNA and trisomy DNA, respectively. The difference between these two groups was highly significant (P < 0.001). These results indicate that this quantitative method is practical and may be used for the prenatal diagnosis of Down's syndrome caused by trisomy 21.

Cells, Cultured↗
PubMed2002

Evaluation of mixed hematopoietic chimerism in pediatric patients with leukemia after allogeneic stem cell transplantation by quantitative PCR analysis of variable number of tandem repeat and testis determination gene.

In order to monitor the clinical outcome of pediatric patients with leukemia following allogeneic hematopoietic transplantation, tests of variable number of tandem repeat (VNTR) and sex determination by quantitative polymerase chain reaction (PCR) were performed. PCR results combined with the blast counts from 21 leukemia patients were analyzed. Complete chimerism (100% donor cells) was found in 15 cases with remission, and incomplete chimerism in six cases with relapse. In the majority of cases, complete chimerism was always associated with no detectable blasts, while blasts were often detected in association with incomplete chimerism. There is significant correlation (P<0.0001) between the percentage of donor DNA and blast percentage in these patients. Early detection of incomplete chimerism may therefore predict a poor prognosis. In one patient (case 15), a differing percentage of donor DNA was observed between samples of bone marrow and peripheral blood collected on the same day. This may be due to the fact that allogeneic stem cells proliferate at different rates depending on their environment (bone marrow or peripheral blood). In addition, 100% donor cells found in the peripheral blood may not reflect the number of cells in the bone marrow. In case 17, asynchronous engraftment of donor cells was present between the white and red blood cell lineages, indicating that the degree of chimerism may not be the same in all cell lineages. At the time of this report, the significance of this observation is unknown and needs further investigation.

Adolescent↗
PubMed2004

[Detection of fetal SRY gene in maternal plasma by real-time fluorescence quantitative PCR].

OBJECTIVE: To isolate fetal DNA from maternal plasma and examine its fetal origin. METHODS: Fetal DNA in maternal plasma was isolated from 150 samples in the first trimester and mid-trimester of pregnancy, respectively. Real-time fluorescence quantitative polymerase chain reaction PCR (FQ-PCR) was used to determine sex-determining region Y (SRY) gene on Y chromosome. RESULTS: Eighty-two women in the first trimester and 90 women in the mid-trimester carried male fetuses,70 and 90 samples of them were positive, respectively. The mean concentrations were (58.82+/-20.90) copies/ml and (152.08+/-62.61) copies/ml. The results of FQ-PCR were negative in the women who carried female fetuses. CONCLUSION: The results show that fetal SRY gene can be found at a time as early as 42 days of gestation in maternal plasma by the use of FQ-PCR. The number of fetal DNA increases with gestational age. The real-time FQ-PCR is of great value in the non-invasive prenatal diagnosis.

Adult↗
PubMed2001

Real-time quantitative PCR analysis for alpha-thalassemia-1 of Southeast Asian type deletion in Taiwan.

Since homozygosity of the alpha-thalassemia-1 of Southeast Asian (SEA) type deletion results in hydrops fetalis, a novel protocol based on the real-time quantitating polymerase chain reaction (PCR) technique has been developed to quantify the intact and aberrant alpha-globin genes in adults. The ratio of the normal/SEA-bearing alpha-globin genes was expressed in cycle threshold (C(T)) values. Theoretically, a relative ratio of one to one was anticipated in individuals carrying the SEA type deletion. Twenty-five heterozygous and 20 normal cases were analyzed retrospectively with this protocol. Data showed that the CT values for the intact alpha-globin gene allele and the allele bearing the SEA type deletion in carriers were 28.74+/-1.49 and 26.46+/-2.05, respectively. Therefore, the ratio of normal/SEA type deletion-bearing alpha-globin genes in the carriers was 1.09+/-0.043. No ambiguous results were observed from other less common genotypes associated with alpha-thalassemia, such as the Philippine type deletion. Based on the results, we concluded that this protocol could provide a rapid method to mass screen carriers with alpha-thalassemia-1 of SEA type deletion in this region.

Alleles↗
PubMed2005

Quantitative PCR of bone marrow BCL2/IgH+ cells at diagnosis predicts treatment response and long-term outcome in follicular non-Hodgkin lymphoma.

By real-time quantitative polymerase chain reaction (RQ-PCR), we evaluated BCL2/IgH(+) cells in the bone marrow (BM) and peripheral blood (PB) from 86 patients with follicular lymphoma treated with the sequential administration of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) and rituximab. At diagnosis, the amount of BCL2/IgH(+) cells in the BM was low (1 BCL2/IgH(+) cell in 1000-100 000 normal cells) in 43% of patients, intermediate (1 in 100-1000) in 34%, and high (> 1 in 100) in 23%. A 2 log decrease of BCL2/IgH(+) cells was achieved after CHOP and an additional 2 log reduction following rituximab. By multivariate analysis, a low level of BCL2/IgH(+) cells in the BM at diagnosis was the best predictor for the achievement of a complete clinical and molecular response. At 5 years, the event-free survival rates of patients with a low/intermediate or high tumor infiltration in the BM were 59% and 32%, respectively. The freedom from recurrence of patients who achieved a molecular response in the BM, no matter whether after CHOP alone or CHOP and rituximab, was 64% as compared to 32% for patients who did not (P < .006). RQ-PCR performed at presentation on BM samples predicts treatment response and long-term clinical outcome in patients with follicular lymphoma.

Adult↗
PubMed2006

Quantitative PCR targeting 16S rRNA and reductive dehalogenase genes simultaneously monitors multiple Dehalococcoides strains.

The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria- and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.

Bacterial Typing Techniques↗
PubMed2003

Development and clinical application of a fully controlled quantitative PCR assay for cell-free cytomegalovirus in human plasma.

BACKGROUND: Cytomegalovirus (HCMV) disease continues to be a major problem in certain patient groups, including bone marrow transplant (BMT) recipients. The quantification of HCMV genome is clinically useful for the diagnosis of HCMV disease, for the virological surveillance of high-risk patients and for monitoring antiviral therapy. OBJECTIVES: To develop a novel, robust, and fully controlled PCR (qPCR) for the quantification of HCMV DNA in plasma samples and to demonstrate its clinical usefulness in the BMT setting. STUDY DESIGN: The newly developed HCMV qPCR employs cell culture-derived murine CMV as an internal control for both extraction and amplification. Following amplification using common primers, detection of both internal control and patient HCMV amplicons is by specific probes and a chemiluminescence microtitre plate system. Its performance was evaluated using the routine non-quantitative nested HCMV PCR on whole blood (NQPCR) and correlated with clinical events such as disease and antiviral therapy. RESULTS: A high level of concordance (85.1%) was found between the novel assay and the NQPCR, with the qPCR being slightly more sensitive. The samples giving discordant results generally had levels of HCMV DNA close to the limit of detectability or had been stored for prolonged periods. CONCLUSIONS: The use of plasma as an analyte by the newly developed assay avoids the detection of cell-associated virus. On the other hand, testing a comparatively large volume of plasma ensures that sensitivity is not compromised by not detecting cell-associated HCMV. In a small preliminary evaluation in BMT recipients, changes in HCMV 'viral load' correlated with initiation and discontinuation of antiviral therapy and were biologically plausible.

Base Sequence↗
PubMed2004

Measurement of relative copy number of CDKN2A/ARF and CDKN2B in bladder cancer by real-time quantitative PCR and multiplex ligation-dependent probe amplification.

Many tumors have large homozygous deletions of the CDKN2A locus (encoding p14(ARF) and p16) and of CDKN2B (p15). Our aim was to determine which gene is the major target in bladder cancer. We used quantitative real-time PCR (RTQ-PCR) to determine copy number of p15, of p14(ARF) exon 1beta, and p16 exon 2 in 22 tumor cell lines and 83 bladder tumors, some of which had been assessed previously by duplex PCR. Titration experiments showed that homozygous deletion could be detected in the presence of up to 30% normal DNA. Results for cell lines were compatible with previous cytogenetic analyses. Ten cell lines and 32 tumors (38.5%) had homozygous deletion of at least one target. Thirteen tumors (15.7%) had deletion of all three targets. Two tumors had deletion of p14(ARF) exon 1beta alone and four of p16 exon 2 alone. RTQ-PCR detected more homozygous deletions than duplex PCR. Finally we used a multiplex ligation-dependent probe amplification kit to provide independent confirmation of results. We conclude that with appropriate controls RTQ-PCR is a sensitive and robust method to detect copy number changes in tumors even in the presence of contaminating normal cell DNA.

Cell Cycle Proteins↗
PubMed2005

Real-time quantitative PCR analysis of regions involved in gene amplification reveals gene overdose in low-grade astrocytic gliomas.

We have studied gene amplification of genes located in 1q32 (GAC1, ELF3, MDM4, and ren1), 4q11 (PDGFR-alpha), and in 12q13-14 (MDM2 and CDK4) using quantitative real-time PCR in a group of 86 tumors consisting of 44 WHO grade IV glioblastomas (GBM) (34 primary and 10 secondary tumors), 21 WHO grade III anaplastic astrocytomas (AA), and 21 WHO grade II astrocytomas (AII). Gene amplification was present in 56 of the 86 samples (65%) in at least 1 gene in our series. GAC1 (51%) and MDM4 (27%) were the most frequently amplified genes within the 1q32 amplicon, and their higher amplification frequency was statistically significant (P<0.05, chi) in the low-grade astrocytomas. Concordant co-amplification was determined for ELF3 and ren1 or ren1 and MDM4 in the grade III-IV tumors. MDM2 amplification was significantly more frequent in primary GBM (16%) than was in secondary GBM (0%). The present study shows that gene amplification in the studied regions is already present in low-grade astrocytic tumors and that amplification of some genes may represent another molecular marker to differentiate primary from secondary GBM.

Astrocytoma↗
PubMed2004

Real-time PCR quantitation of glucocorticoid receptor alpha isoform.

BACKGROUND: The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRalpha) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision. RESULTS: Standard-curves were constructed by employing standardized Jurkat cell culture procedures, both for GRalpha and BCR (breakpoint cluster region), as a normalizing gene. We evaluated standard-curves using five different sets of cell culture passages, RNA extraction, reverse transcription, and qrt-PCR quantification. Intra-assay precision was evaluated using 12 replicates of each gene, for 2 patients, in a single experiment. Inter-assay precision was evaluated on 8 experiments, using duplicate tests of each gene for two patients. Standard-curves were reproducible, with CV (coefficient of variation) of less than 11%, and Pearson correlation coefficients above 0,990 for most comparisons. Intra-assay and inter-assay were 2% and 7%, respectively. CONCLUSION: This is the first method for quantitation of GRalpha expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way.

Adult↗
PubMed2001

Following the course of human leptospirosis: evidence of a critical threshold for the vital prognosis using a quantitative PCR assay.

In order to follow the course of acute human leptospirosis, an ELISA microtiter plate hybridization method was developed for the quantitative determination of Leptospira spp. in biological samples after PCR. The biotin-labelled amplified product (331 bp from the rrs gene) was hybridized with a complementary capture probe covalently linked onto aminated polystyrene wells, and detected using a colorimetric reaction. The mean detection limit was 50 copies per 10 microl. In a prospective study of human leptospirosis cases, we obtained evidence that a density of 10(4) leptospires per ml of blood is a critical threshold for the vital prognosis of the patients. The practicability of the method makes it suitable for use in tropical areas for multicentric studies. Such studies could lead to a better knowledge of the natural history of the human disease. The method is also suitable for experimental evaluation of improved antibiotic treatments for leptospirosis.

Adolescent↗
PubMed1999

Quantitative PCR analysis of c-erb B-2 (HER2/neu) gene amplification and comparison with p185(HER2/neu) protein expression in breast cancer drill biopsies.

A PCR assay using capillary electrophoresis was designed for the detection of c-erbB-2 gene amplification in alcohol-formalin-acetic acid (AFA)-fixed, paraffin-embedded biopsies from 81 consecutive breast tumors. c-erbB-2 expression was analyzed in the same samples using immuno-histochemistry (IHC). In the competitive PCR assay, a single pTag plasmid containing a 4-nucleotide (nt)-deleted copy of a 124-nt sequence of c-erbB-2 and a 4-nt-deleted copy of a 120-nt sequence of GAPDH was co-amplified with genomic DNA extracted from 3 10-micrometer-thick tissue sections of the tumor biopsy. The percentage of tumor cells in the biopsy specimen and the percentage of tumor cells stained with the membrane anti-c-erbB-2 monoclonal antibody CB11 were recorded by a single pathologist on 2 consecutive sections. Among 81 consecutive tumor biopsies assayed by PCR, 21 (26%) displayed unequivocal c-erbB-2 amplification (actual gene copy number, AGCN > 4), 47 (58%) displayed no c-erbB-2 amplification (AGCN </= 2) and 7 (9%) could not be analyzed due to an insufficient amount of DNA. Six samples (7%) were considered inconclusive since the percentage of tumor cells was <20%. Analysis of c-erbB-2 expression by IHC showed that among the 21 amplified specimens 15 displayed strong staining, while all non-amplified samples (47) displayed no or only weak staining. The concordance of the 2 techniques was 91%. We conclude that c-erbB-2 gene amplification can be accurately quantitated by competitive PCR performed on small, fixed and embedded tumor samples.

Adenocarcinoma↗
PubMed2005

Real-time reverse transcription-PCR quantitation of substance P receptor (NK-1R) mRNA.

The substance P (SP)-preferring receptor, neurokinin-1 receptor (NK-1R), has an important role in inflammation, immune regulation, and viral infection. We applied a newly developed real-time reverse transcription (RT)-PCR assay to quantify NK-1R mRNA in human neuronal cell line (NT-2N), a human B-cell line (IM9), monocyte-derived macrophages (MDM), peripheral blood lymphocytes (PBL), and human astroglioma cells (U87 MG). The NK-1R real-time RT-PCR assay has a sensitivity of 100 mRNA copies, with a dynamic range of detection between 10(2) and 10(7) copies of NK-1R gene transcripts per reaction. This assay is highly reproducible, with an intraassay coefficient variation of threshold cycle (Ct) of less than 1.9%. The NK-1R real-time RT-PCR is highly sensitive for quantitative determination of NK-1R mRNA in human immune cells (MDM and PBL) that express low levels of NK-1R mRNA. In addition, the assay has the ability to accurately quantitate the dynamic changes in NK-1R mRNA expression in interleukin-1beta-stimulated U87 MG. These data indicate that the NK-1R real-time RT-PCR has potential for a wide application in investigation of NK-1R expression at the mRNA level under physiological and pathological conditions in both the central nervous system and the immune system.

Cells, Cultured↗
PubMed2002

A simple single-step method for the synthesis of recombinant non-homologous competitor cDNA and its implications for quantitative PCR in biological sciences and molecular diagnostics.

In recent years there has been great interest in quantitative polymerase chain reaction. Consequently, a large number of assays have been developed, of which the one using non-homologous competitors is arguably the most precise. Despite widespread applications, currently there is no simple method to synthesize such competitors. Here a facile and cost-effective, single-step method for synthesis of recombinant, non-homologous, competitor complementary DNA is described. The method can be adapted to generate competitors of any size and sequence. The entire procedure is quick, straightforward and does not require any specialized equipment except a standard thermocycler.

Animals↗
PubMed2006

Development of quantitative PCR methods to analyse neural progenitor cell culture state.

Stem cells have significant potential for tissue engineering and regeneration, and neural stem and progenitor cells have proven promising for neuroregeneration in numerous animal disease and injury models. However, improved approaches must be developed to culture, expand and control the cells. Therefore the development of enhanced methods to quantify cell differentiation would significantly aid both in the basic investigation of cell-fate control mechanisms and in the optimization and validation of cell culture and expansion conditions. Quantitative reverse transcription-PCR methods were developed to quantify cell differentiation state by monitoring the expression of several cell-lineage-specific markers. These methods provide more rapid and readily quantitative results when compared with immunostaining. These methods were also applied in a preliminary investigation of cell-culture conditions, and it was found that regular feeding of cells with fresh medium is necessary to maintain them in an undifferentiated and highly proliferative state. The present study may aid both basic efforts to study the control of neural stem and progenitor differentiation as well as endeavours to optimize cell culture and expansion conditions for biomedical applications.

Base Sequence↗
PubMed2004

Real-time PCR quantitation of clostridia in feces of autistic children.

Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C. bolteae, in stool specimens. Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (CT) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C. bolteae could be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C. bolteae and the following Clostridium groups were statistically significant: mean counts of C. bolteae and clusters I and XI in autistic children were 46-fold (P = 0.01), 9.0-fold (P = 0.014), and 3.5-fold (P = 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 x 10(8) CFU/g in autistic children and 4.8 x 10(8) CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract.

Autistic Disorder↗