SEARCH · Search PubMed

Results for “Quantitative PCR”

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.

SourceResearch connectionFrom yearThrough year

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

At least 1,405 records · Page 78Linked to original sources

PubMed2004

Twelve hour real-time PCR technique for the sensitive and specific detection of Salmonella in raw and ready-to-eat meat products.

Rapid pathogen testing is vital to the food industry. Enzyme immunoassays (EIA) provide reliable negative results in 48 h, but a presumptive positive (suspect) EIA result must be confirmed by traditional culture methods, requiring an additional 72 h. Polymerase chain reaction (PCR) testing technology is accepted as an accurate diagnostic tool. However, traditional PCR techniques can require several days. We sought to develop a rapid, real-time quantitative PCR technique for detecting Salmonella spp. in food products. Salmonella spp. was inoculated into raw and ready-to-eat beef products. Total DNA was extracted and used as template for PCR amplification in the LightCycler (Roche Diagnostics Corp., Idaho Technology Inc., Idaho Falls, ID) PCR instrument. Salmonella-specific PCR primers were designed to amplify a 251 base pair product from the junction of SipB and SipC. Fluorescently-labeled hybridization probes were designed to anneal to SipB and SipC. Salmonella was detected down to 1 colony forming unit/ml in food products. The results of real-time PCR correlated 100% to those of visual immunoprecipitate and culture. PCR methods using the LightCycler can detect and confirm the presence or absence of Salmonella spp. in raw and ready-to-eat beef products within 12 h with increased sensitivity compared to traditional culture and EIA methods.

Animals↗

PubMed2006

Genetic characterization of strains of Saccharomycescerevisiae responsible for 'refermentation' in Botrytis-affected wines.

AIMS: Saccharomyces cerevisiae is responsible for alcoholic fermentation of wines. However, some strains can also spoil sweet Botrytis-affected wines. Three 'refermentation' strains were isolated during maturation. Characterization of those strains in regards to their fingerprint, rDNA sequence and resistance to SO2, which constituted the main source of stress in Botrytis-affected wines, was carried out. METHODS AND RESULTS: Refermentation strains could be clearly discriminated by interdelta fingerprinting. However, they exhibited close relationships by karyotyping. A part of RDN1 locus sequence was examined by using PCR-RFLP and PCR-DGGE. The resistance of refermentation strains to SO2 was performed by using real time quantitative PCR focusing on SSU1 gene. CONCLUSIONS: Results suggested that refermentation strains were heterozygote in 26S rDNA and their ITS1-5.8S rDNA-ITS2 region sequence revealed relationships with 'flor' strains. As described in the literature for flor strain, two out of three refermentation strains constitutively developed a higher level of SSU1 expression than the reference strains, improving their putative tolerance to SO2. Therefore, refermentation strains of S. cerevisiae had developed many strategies to survive during maturing sweet wines. SIGNIFICANCE AND IMPACT OF THE STUDY: Singularities in rDNA sequence and SSU1 overexpression revealed a natural adaptation. Moreover, genomic relationship between flor and refermentation strains suggested that stress sources could induced selection of survivor strains.

Acetaldehyde↗

PubMed2005

HHV-6-related acute liver failure in two immunocompetent adults: favourable outcome after liver transplantation and/or ganciclovir therapy.

Fulminant hepatitis of unknown origin remain a significant cause of mortality, for which liver transplantation is often considered as the only therapeutic option. In retrospective studies, human herpesvirus 6 (HHV-6) infections have been associated with such diseases, but the diagnosis of HHV-6 infection of the liver is rarely established during the acute phase of liver failure. Using real-time polymerase chain reaction (PCR), we diagnosed two cases of severe acute liver failure (ALF) related to HHV-6 occurring in immunocompetent young adults. Both cases had a favourable outcome, one after valganciclovir therapy, one after liver transplantation associated with ganciclovir. Viral origin was evidenced in each case by the detection of high amounts of HHV-6 DNA in liver tissue by the PCR assay. The decrease of intrahepatic viral load after therapeutic intervention was also monitored by quantitative PCR and paralleled in the two cases the clinical improvement. Diagnosis of HHV-6 infection must be systematically evoked in case of unexplained ALF, since it might lead to specific therapeutic interventions, in addition of liver transplantation.

Administration, Oral↗

PubMed2001

Linear mixture analysis: a mathematical approach to resolving mixed DNA samples.

With the advent of PCR-based STR typing systems, mixed samples can be separated into their individual DNA profiles. Quantitative peak information can help in this analysis. However, despite such advances, forensic mixture analysis still remains a laborious art, with the high cost and effort often precluding timely reporting. We introduce here a new automated approach to resolving forensic DNA mixtures. Our linear mixture analysis (LMA) is a straightforward mathematical approach that can integrate all the quantitative PCR data into a single rapid computation. LMA has application to diverse mixture problems. As demonstrated here on laboratory STR data, LMA can assess the quality and utility of its solutions. Such rapid and robust methods for computer-based analysis of DNA mixtures may help in reducing crime.

Algorithms↗

PubMedDate not supplied

Maintained cellular function of adrenal medullary cells in parkinsonian dysautonomia.

Adrenal gland involvement in Parkinson's disease was reported by different authors. Further studies became relevant after adrenal was proposed as dopaminergic donor for neurotransplantation. Chromaffin cells were grown in culture and the effects of nerve growth factor (NGF) tested: no differences were observed between parkinsonian and control cells. The expression of the beta-NGF mRNA in the parkinsonian adrenal was analyzed: a specific cDNA was synthesized and a 168 bp portion amplified using PCR. The products were identified and the identity of the fragment was confirmed by sequencing. Quantitative PCR demonstrated a beta-NGF mRNA concentration exceeding 5 fg/micrograms of total adrenal RNA. These findings demonstrate the retained functional capacity of the parkinsonian adrenal to respond to NGF and express the beta-NGF mRNA.

Adrenal Medulla↗

PubMed2006

Early detection of Biscogniauxia nummularia in symptomless European beech (Fagus sylvatica L.) by TaqMan quantitative real-time PCR.

AIMS: To develop a quantitative real-time PCR (Rt PCR) assay for the early detection of Biscogniauxia nummularia, a xylariaceous fungus that causes strip-canker and wood decay on European beech (Fagus sylvatica L.). METHODS AND RESULTS: The molecular assay was based on TaqMan chemistry using species-specific primers and a fluorogenic probe designed on the ITS1 sequence of rRNA gene clusters. The specificity of the oligonucleotides and the probe were tested using the DNA of B. nummularia isolates from different geographic areas, of phylogenetically related species, and of some fungi commonly colonizing European beech bark and wood. A total of 31 symptomless and symptomatic shoots of European beech were collected from three forest sites in the Apennine Mountains of Italy. The percentage of positive detections of B. nummularia with the TaqMan assay was 78.6%, compared with only 14.3% of positive isolations on growth media for two sites. CONCLUSIONS: In shoots, the quantitative Rt PCR assay detected down to 8.0-fg fungal DNA per microgram of total DNA extracted. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed in quantitative Rt PCR, by using TaqMan chemistry, revealed a rapid and sensitive method useful for the early detection of B. nummularia in symptomless European beech twigs.

DNA Primers↗

PubMed2004

Measurement of the antibiotic susceptibility of Coxiella burnetii using real time PCR.

The objective of this study was to determine MICs of antibiotics for two reference strains of Coxiella burnetii using real time quantitative PCR. The method was very sensitive and specific and allowed the evaluation of the doubling time of Nine Mile and Q212 strains: 37 and 15 h, respectively. Dose response curves of antibiotics were used to determine MICs. Those of doxycycline, fluoroquinolone compounds and rifampicin were in the range 1-4 mg/l. Telithromycin was the most effective macrolide compound with MICs of 1-2 mg/l. The results confirmed previous reports on the accuracy of this new method for the determination of the antibiotic susceptibility of C. burnetii and could be used for the screening of new drugs.

Animals↗

PubMed1995

Analysis of TGF-beta 1 gene expression in contused rat spinal cord using quantitative RT-PCR.

We have used northern blot analysis and quantitative reverse transcription polymerase chain reaction (RT-PCR) to determine the postinjury expression profile of the transforming growth factor-beta 1 (TGF-beta 1) gene in the contused rat spinal cord. Spectrophotometric estimates of total sample RNA and quantitative analyses of cyclophilin mRNA using RT-PCR served as controls for comparisons between samples. No changes in cyclophilin gene expression were found at any postinjury survival times. The results of the TGF-beta 1 analyses, which were carried out on spinal cord samples taken at postinjury intervals ranging from 6 h to 10 days, show that the amount of TGF-beta 1 mRNA present in spinal cord increases rapidly following injury, reaching maximum levels 7 days postinjury. Unoperated control samples contained approximately 2 x 10(8) molecules of TGF-beta 1 mRNA/0.5 microgram total RNA. By 1 day postinjury, the amount of TGF-beta 1 mRNA in the cord had increased by a factor of 2.5 to 5 x 10(8) molecules/0.5 microgram total RNA. At 7 days postinjury, there were approximately 15 x 10(8) molecules of TGF-beta 1 mRNA/0.5 microgram total RNA. By 10 days postinjury the amount of TGF-beta 1 mRNA present in the spinal cord had declined to 8 x 10(8) molecules of TGF-beta 1 mRNA/0.5 microgram total RNA, a value similar to that observed at 3 days postinjury. The roles that TGF-beta 1 might play in modifying cellular responses in injured spinal cord are discussed.

Animals↗

PubMed2006

The effect of a novel recombination between the homeobox gene NKX2-5 and the TRD locus in T-cell acute lymphoblastic leukemia on activation of the NKX2-5 gene.

BACKGROUND AND OBJECTIVES: The NK-like homeobox gene (NKX2-5/CSX) plays a crucial role in cardiac development but is not normally expressed in hematopoietic cells. Here, we describe for the first time a fusion between NKX2-5 and the T-cell receptor delta locus (TRD) resulting in NKX2-5 activation in a case of T-cell acute lymphoblastic leukemia (T-ALL). DESIGN AND METHODS: Genomic DNA from a T-ALL patient with an atypical rearrangement, detected by Southern blotting, was analyzed by ligation-mediated polymerase chain reaction (PCR) with TRD-specific primers. Expression of NKX2-5 was analyzed by real-time quantitative PCR in the T-ALL case with the NKX2-5-TRD rearrangement, 18 other cases of T-ALL, three T-ALL derived cell lines, two non-hematopoietic cell lines, peripheral blood mononuclear cells from six healthy individuals and sorted thymocyte subsets. RESULTS: Sequence analysis of ligation-mediated PCR products revealed a novel rearrangement between the third diversity segment of the TRD locus (TRDD3) and a region on chromosome 5q35.1 located 32 kb downstream of the NKX2-5/CSX gene. As a result of this recombination NKX2-5 was placed under influence of the TRD enhancer, resulting in strong ectopic NKX2-5 expression. High NKX2-5 expression was also found in the T-cell lines PEER and CCRF-CEM, which harbor an NKX2-5-BCL11B rearrangement, and in the embryonic kidney cell line 293. NKX2-5 was not expressed in any of the major thymocyte subsets, in normal peripheral blood mononuclear cells, or in the majority (17/18) of the other cases of T-ALL. INTERPRETATION AND CONCLUSIONS: Our finding of overexpression of yet another homeobox gene in T-ALL further supports the hypothesis that homeobox genes play an important role in malignant transformation of particular types of T-ALL.

Base Sequence↗

PubMed2004

Molecular characterization of conditionally immortalized cell lines derived from mouse early embryonic inner ear.

Inner ear sensory hair cells (HCs), supporting cells (SCs), and sensory neurons (SNs) are hypothesized to develop from common progenitors in the early embryonic otocyst. Because little is known about the molecular signals that control this lineage specification, we derived a model system of early otic development: conditionally immortalized otocyst (IMO) cell lines from the embryonic day 9.5 Immortomouse. This age is the earliest stage at which the otocyst can easily be separated from surrounding mesenchymal, nervous system, and epithelial cells. At 9.5 days post coitum, there are still pluripotent cells in the otocyst, allowing for the eventual identification of both SN and HC precursors--and possibly an elusive inner ear stem cell. Cell lines derived from primitive precursor cells can also be used as blank canvases for transfections of genes that can affect lineage decisions as the cells differentiate. It is important, therefore, to characterize the "baseline state" of these cell lines in as much detail as possible. We characterized seven representative "precursor-like" IMO cell populations and the uncloned IMO cells, before cell sorting, at the molecular level by polymerase chain reaction (PCR) and immunocytochemistry (IHC), and one line (IMO-2B1) in detail by real-time quantitative PCR and IHC. Many of the phenotypic markers characteristic of differentiated HCs or SCs were detected in IMO-2B1 proliferating cells, as well as during differentiation for up to 30 days in culture. These IMO cell lines represent a unique model system for studying early stages of inner ear development and determining the consequences of affecting key molecular events in their differentiation.

Animals↗

PubMed2005

Epidemiologic and viral factors associated with cervical neoplasia in HPV-16-positive women.

While infection with high-risk HPV is the most important risk factor for cervical cancer, HPV alone is insufficient. Our purpose was to identify viral and epidemiologic factors associated with cervical disease in HPV-16 DNA-positive women referred to colposcopy. We used a standardized interview to collect epidemiologic data from consenting women. Total nucleic acids from exfoliated cervical cells were used for all viral assays (HPV detection and typing using L1 consensus PCR with line probe hybridization, variant classification by sequencing, viral load and transcript copy determination by quantitative PCR and transcript pattern by nested RT-PCR). Cervical disease was based on colposcopic biopsy. Logistic regression was used to calculate ORs with 95% CIs. There were 115 HPV-16 positive women among 839 enrollees. By univariate analyses, age >25 years (OR = 3.05, 95% CI 1.20-7.76), smoking (OR = 3.0, 95% CI 1.19-7.56), high viral load (OR = 5.27, 95% CI 2.05-13.60), detection of both E6 and E6*I transcripts (OR = 10.0, 95% CI 2.1-47.58) and high transcript copies (OR = 5.56, 95% CI 2.05-13.60) were significant risk factors for CIN III with reference to No CIN/CIN I. Less than a third of the women (31.5%) had prototype HPV-16 detected, and variants showed no association with disease, viral load or transcription. Viral DNA and transcript copies were highly correlated, and the ratio of transcript copies to DNA copies was not changed with disease status. While viral load, transcript copies and transcript pattern were statistically associated with CIN III, none of these measures effectively discriminated between HPV-16 women with disease requiring treatment and those who could be followed. Cellular proliferation and differentiation pathways affected by HPV should be investigated as biomarkers for cervical cancer screening.

Adolescent↗

PubMed1996

Detection of minimal residual disease in all: biology, methods, and applications.

The PCR technique appears to be the most sensitive method for detecting residual disease in ALL and can be applied to a high percentage of cases by amplifying sequences of the antigen-receptor genes. The PCR studies to date suggest that this sensitive technique can detect residual disease in virtually all patients during the first year of treatment. The residual disease becomes undetectable in the majority of patients by the end of treatment; however, a subset of patients remain PCR positive at a time when therapy is electively discontinued. The development of a highly accurate quantitative PCR technique may allow the possibility of distinguishing the patterns of residual disease for patients who will be cured by treatment from those who relapse. If such a pattern can be discerned, then an immediate benefit for PCR monitoring will be that clinicians will have the opportunity to test whether treating patients at the time of 'molecular relapse' will help to improve the cure rate for this disease. The PCR studies of remission marrows at the end of treatment raise a number of questions about the biology of disease persistence in patients who remain in extended 'remission.' A commitment to obtaining and analyzing bone marrow specimens in patients who have completed therapy is necessary to discern whether novel strategies, such as immunomodulatory manipulations, are needed to control or eradicated residual disease in patients who have completed planned chemotherapy. Thus, the long-term benefit of residual disease monitoring by PCR may be a better understanding of the biology and definition of 'cure' in ALL.

Child↗

PubMed2005

Development of a rapid detection and enumeration method for thermophilic bacilli in milk powders.

Thermophilic strains of Geobacillus, Anoxybacillus and Bacillus that are able to grow at 55 degrees C and above are recognized as commonly occurring contaminants during the production of milk powders. In particular, Anoxybacillus flavithermus strain C and Bacillus licheniformis strain F are often the most prevalent. We describe here the development of a TaqMan-based real-time-PCR assay using a small amplicon of the ribosomal 16S rRNA gene for the selective and quantitative detection of thermophilic bacilli in milk powders. We further present an effective, rapid and inexpensive method for the isolation of total bacterial DNA from milk powder for quantitative PCR analysis within 20 min. With this method, the detection of thermophilic bacilli in milk powder can be accomplished within 1 h. The detection limit for reconstituted and inoculated milk was 8 vegetative cfu ml(-1) and 64 spores ml(-1), respectively.

Animals↗

PubMedDate not supplied

Quantification of human astroviruses in sewage using real-time RT-PCR.

Human astroviruses constitute a significant cause of acute diarrhea in children. Viral transmission occurs via the fecal-oral route, predominantly person-to-person, but the consumption of fecally contaminated water and shellfish has also been implicated. Viral pathogen detection in water, especially, in wastewater, is difficult and PCR is widely used to detect these viruses. Despite the recent development of real-time quantitative PCR, quantification of astroviruses in sewage had not been available up to now. We have developed a method to quantify astroviruses in sewage. For this purpose, we designed a set of primers and a fluorogenic probe located at the 3' -end of the genome of human astroviruses. The amplified region was cloned and the plasmid was transcribed to generate calibration standards for quantification. After validation of the standards, the method was evaluated in artificially contaminated samples. To validate the method on naturally contaminated samples, raw and treated wastewater samples were collected monthly for one year in a sewage treatment plant. Astrovirus genomes were detected in all samples collected at the entrance to the sewage treatment plant, with a mean value of 4.1 x 10(6) astrovirus genomes for 100 ml. Effluents were less strongly contaminated, with a mean value of 1.01 x 10(4) astrovirus genomes. The high prevalence of astroviruses in sewage treatment plant effluents indicates that these plants are not efficiently eliminating the virus. This is a major public health concern and new techniques of depuration are needed. Our method could be effectively used in evaluating new treatment processes to reduce the viral load in the effluent of treatment plants.

Environmental Monitoring↗

PubMed2002

A novel, essential control for clonality analysis with human androgen receptor gene polymerase chain reaction.

The most widely used technique for determining clonality based on X-chromosome inactivation is the human androgen receptor gene polymerase chain reaction (PCR). The reliability of this assay depends critically on the digestion of DNA before PCR with the methylation-sensitive restriction enzyme HpaII. We have developed a novel method for quantitatively monitoring the HpaII digestion in individual samples. Using real-time quantitative PCR we measured the efficiency of HpaII digestion by measuring the amplification of a gene that escapes X-chromosome inactivation (XE169) before and after digestion. This method was tested in blood samples from 30 individuals: 2 healthy donors and 28 patients with myelodysplastic syndrome. We found a lack of XE169 DNA reduction after digestion in the granulocytes of two myelodysplastic syndrome patients leading to a false polyclonal X-chromosome inactivation pattern. In all other samples a significant reduction of XE169 DNA was observed after HpaII digestion. The median reduction was 220-fold, ranging from a 9.0-fold to a 57,000-fold reduction. Also paraffin-embedded malignant tissue was investigated from two samples of patients with mantle cell lymphoma and two samples of patients with colon carcinoma. In three of these cases inefficient HpaII digestion led to inaccurate X-chromosome inactivation pattern ratios. We conclude that monitoring the efficiency of the HpaII digestion in a human androgen receptor gene PCR setting is both necessary and feasible.

DNA↗

PubMed2001

The mRNA level of the potassium-chloride cotransporter KCC2 covaries with seizure susceptibility in inferior colliculus of the post-ischemic audiogenic seizure-prone rat.

Cardiac arrest and resuscitation were used to induce brain damage and susceptibility to sound-triggered seizures in Sprague-Dawley rats. Glucose preloading was used to vary seizure susceptibility. Because loop diuretics can block these seizures, we investigated changes in KCC2, a potassium-chloride cotransporter, in the inferior colliculus - the origin of the seizures. Using polymerase chain reaction (PCR), we found that collicular KCC2 mRNA levels covaried with seizure susceptibility in these animals. Using quantitative PCR, we found that a fivefold increase in collicular KCC2 mRNA levels was associated with a doubling of seizure incidence. A hypothesis linking KCC2 activity to seizure susceptibility is presented.

Animals↗

PubMed2000

Large-scale screening for human parvovirus B19 DNA by PCR: application to the quality control of plasma for fractionation.

BACKGROUND AND OBJECTIVES: Because human parvovirus B19 (B19) has been transmitted by various plasma-derived medicinal products, the 'Laboratoire français du Fractionnement et des Biotechnologies' (LFB) implemented PCR screening of plasma pool samples for B19 DNA as part of the quality control of plasma source material. MATERIALS AND METHODS: Plasma pool samples (average of 46.5 donations) were tested for B19 DNA by PCR and by immunological detection of PCR products. The viral DNA content was determined by means of a TaqMantrade mark-based, quantitative PCR. RESULTS: From plasma corresponding to 2-year collections in France, and representing 4.26 million donations from approximately 1.25 million voluntary unpaid donors, the average frequency of positive donations was 1/5,950 and reached 1/1,420 during an epidemic. Levels of B19 DNA in positive pools ranged from <10(2) to 10(11) copies/ml. CONCLUSION: A large-scale PCR plasma screening increased the safety of LFB's wide range of products with respect to B19, a virus particularly resistant to physicochemical inactivation procedures.

Consumer Product Safety↗

PubMed2006

In vivo distribution of human adipose-derived mesenchymal stem cells in novel xenotransplantation models.

The potential for human adipose-derived mesenchymal stem cells (AMSC) to traffic into various tissue compartments was examined using three murine xenotransplantation models: nonobese diabetic/severe combined immunodeficient (NOD/SCID), nude/NOD/SCID, and NOD/SCID/MPSVII mice. Enhanced green fluorescent protein was introduced into purified AMSC via retroviral vectors to assist in identification of cells after transplantation. Transduced cells were administered to sublethally irradiated immune-deficient mice through i.v., intraperitoneal, or subcutaneous injection. Up to 75 days after transplantation, tissues were harvested and DNA polymerase chain reaction (PCR) was performed for specific vector sequences as well as for human Alu repeat sequences. Duplex quantitative PCR using human beta-globin and murine rapsyn primers assessed the contribution of human cells to each tissue. The use of the novel NOD/SCID/MPSVII mouse as a recipient allowed rapid identification of human cells in the murine tissues, using an enzyme reaction that was independent of surface protein expression or transduction with an exogenous transgene. For up to 75 days after transplantation, donor-derived cells were observed in multiple tissues, consistently across the various administration routes and independent of transduction parameters. Tissue localization studies showed that the primary MSC did not proliferate extensively at the sites of lodgement. We conclude that human AMSC represent a population of stem cells with a ubiquitous pattern of tissue distribution after administration. AMSC are easily obtained and highly amenable to current transduction protocols for retroviral transduction, making them an excellent avenue for cell-based therapies that involve a wide range of end tissue targets.

Adipose Tissue↗