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Results for “Quantitative PCR”

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PubMed2002

Telomere measurement by quantitative PCR.

It has long been presumed impossible to measure telomeres in vertebrate DNA by PCR amplification with oligonucleotide primers designed to hybridize to the TTAGGG and CCCTAA repeats, because only primer dimer-derived products are expected. Here we present a primer pair that eliminates this problem, allowing simple and rapid measurement of telomeres in a closed tube, fluorescence-based assay. This assay will facilitate investigations of the biology of telomeres and the roles they play in the molecular pathophysiology of diseases and aging.

Adolescent↗
PubMed2005

Detection of pathogenic leptospires by real-time quantitative PCR.

Definitive diagnosis of leptospirosis has traditionally depended upon the isolation of leptospires from clinical specimens or the demonstration of seroconversion in paired acute and convalescent serum samples. Both of these approaches require expertise not routinely available in clinical laboratories and usually result in delayed diagnosis. Conventional PCR assays have been developed, but all have limitations which have restricted their widespread use. In order to overcome these limitations, a real-time PCR assay was developed using a 423 bp target on the lipL32 gene, which is conserved among pathogenic serovars of LEPTOSPIRA: Reactions were monitored by SYBR green fluorescence and melting curve analysis. Representative serovars from 16 species of Leptospira and over 40 species of other bacteria and fungi were tested. Positive results were obtained with all pathogenic leptospiral serovars, with the exception of Leptospira fainei serovar Hurstbridge. The analytical sensitivity of this assay was 3 genome equivalents per reaction; approximately 10 genome equivalents were detectable in human urine. Leptospiral DNA was amplified from blood containing EDTA or citrate anticoagulants, but heparin, sodium polyanetholesulfonate and saponin were inhibitory. The assay successfully detected leptospiral DNA from serum and urine samples of patients with leptospirosis. This assay has the potential to facilitate rapid, sensitive diagnosis of acute leptospirosis.

Bacterial Outer Membrane Proteins↗
PubMed1994

A competitive deletion mutant quantitative PCR assay for angiotensin-converting enzyme mRNA in smooth muscle cells.

To quantify angiotensin-converting enzyme (ACE) mRNA, we have developed a reverse transcription (RT)-coupled competitive PCR (RT-PCR) assay with a deletion mutant internal standard. The RT-PCR detects ACE mRNA from both human and bovine sources. ACE mRNA was detected in total RNA from cultured human saphenous vein smooth muscle cells (HuSV-SMCs) and from bovine pulmonary artery (BPA) SMCs. BPA-SMC expressed ninefold less ACE mRNA than BPA endothelial cells, and threefold less than HuSV-SMCs. Apparent amounts of ACE mRNA were 118,350 +/- 2,300 copies in HuSV-SMCs and 42,200 +/- 11,300 copies in BPA-SMCs per microgram of total cell RNA. The accuracy of the absolute values is subject to the limitations of the assumptions used to calculate them. These data support the hypothesis that components of the renin-angiotensin system are transcribed by SMCs.

Animals↗
PubMed2026

Testing for Bacteria and Fungi in Cells and Ancillary Reagents-Probe-Based Quantitative PCR Assays.

This document specifies the technical elements for bacteria and fungi detection by PCR assay which enables rapid, broad-spectrum detection of bacteria and fungi with high sensitivity. Qualitative judgement is based on LOD. Key detection points include efficient nucleic acid extraction, broad-spectrum primer-probe design, high amplification efficiency and minimised background interference.

Editorial↗
PubMed2005

Molecular analysis of the EGFR gene in astrocytic gliomas: mRNA expression, quantitative-PCR analysis of non-homogeneous gene amplification and DNA sequence alterations.

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein with tyrosine kinase activity. This report investigates the presence of mutations, amplification and/or over-expression of the EGFR gene in 86 glial tumours including 44 glioblastomas, 21 anaplastic astrocytomas, and 21 WHO grade II astrocytomas, using polymerase chain reaction/single-strand conformation polymorphism, semiquantitative reverse-transcription-polymerase chain reaction (RT-PCR) and Southern Blot techniques. Gene amplification values were found in 34 tumours. Amplification levels were not uniform, as the transmembrane region presented lower amplification rates than extra- and intracellular domains. For the 19 samples with sufficient available tumour tissue we found over-expression in 11, and no EGFR mRNA expression in three. Ten cases showed deletion transcripts, and EGFR VIII was identified in all of these cases. One of the cases with EGFR vIII also presented a truncated form, C-958, while another showed an in frame tandem duplication of exons 18--25. We found 14 cases with sequence/structure gene alterations, including seven on which genomic novel DNA changes were identified: a missense mutation (1052C > T/Ala265Val), an insertion (InsCCC2498/Ins Pro748), three intronic changes (E6+72delG, E22--14C>G and E18--109T>C), a new polymorphic variant E12+ 22A > T, and one case that presented a 190 bp insertion, that was produced by the intron-7-exon-8 duplication and generated a truncated EGFR with intact exons 1--8 followed by an additional amino acidic sequence: Val-Ile-Met-Trp. These findings corroborate that EGFR is non-randomly involved in malignant glioma development and that different mutant forms participate in aberrant activation of tyrosine kinase pathways.

Astrocytoma↗
PubMed2004

Rapid detection of Oenococcus oeni in wine by real-time quantitative PCR.

AIMS: To develop a real-time polymerase chain reaction (PCR) method for rapid detection and quantification of Oenococcus oeni in wine samples for monitoring malolactic fermentation. METHODS AND RESULTS: Specific primers and fluorogenic probe targeted to the gene encoding the malolactic enzyme of O. oeni were developed and used in real-time PCR assays in order to quantify genomic DNA either from bacterial pure cultures or wine samples. Conventional CFU countings were also performed. The PCR assay confirmed to be specific for O. oeni species and significantly correlated to the conventional plating method both in pure cultures and wine samples (r = 0.902 and 0.96, respectively). CONCLUSIONS: The DNA extraction from wine and the real-time PCR quantification assay, being performed in ca 6 h and allowing several samples to be concurrently processed, provide useful tools for the rapid and direct detection of O. oeni in wine without the necessity for sample plating. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid quantification of O. oeni by a real-time PCR assay can improve the control of malolactic fermentation in wines allowing prompt corrective measures to regulate the bacterial growth.

Colony Count, Microbial↗
PubMed2000

Multiplex and real-time quantitative PCR on fetal DNA in maternal plasma. A comparison with fetal cells isolated from maternal blood.

Fetal DNA has recently been detected in maternal plasma by PCR and has shown promise for the prenatal determination of fetal sex or rhesus D. In order to obtain the maximum amount of information from this fetal genetic material, we have devised a sensitive multiplex PCR method to permit simultaneous analysis for both the SRY locus and the rhesus D gene. Our studies show that this technique is very sensitive and specific. In the 22 cases from rhesus D negative women examined, we were able to determine both fetal genotypes correctly. In the parallel enrichment for fetal cells, fetal erythroblasts were only detected in 14 of the 19 cases. Our data also indicate that fetal DNA from rhesus D positive fetuses is present in maternal plasma even after prophylactic anti-D treatment. Furthermore, since fetal cells have been reported to be elevated in pregnancies with aneuploid fetuses, we have quantified the amount of fetal DNA present in the maternal plasma of 10 such affected pregnancies by real-time PCR. Our results indicate that fetal DNA is elevated under such circumstances when compared to gestationally matched normal pregnancies (mean of 7% in aneuploid samples versus 3.5% in normal pregnancies). These results indicate that the quantification of fetal DNA in maternal plasma may be an additional screening tool for pregnancies at risk of bearing an aneuploid fetus.

DNA↗
PubMed2000

Rapid detection and quantification of members of the archaeal community by quantitative PCR using fluorogenic probes.

We describe a rapid, reproducible, and sensitive method for detection and quantification of archaea in naturally occurring microbial communities. A domain-specific PCR primer set and a domain-specific fluorogenic probe having strong and weak selectivity, respectively, for archaeal rRNA genes (rDNAs) were designed. A universal PCR primer set and a universal fluorogenic probe for both bacterial and archaeal rDNAs were also designed. Using these primers and probes, we demonstrated that detection and quantification of archaeal rDNAs in controlled microbial rDNA assemblages can be successfully achieved. The system which we designed was also able to detect and quantify archaeal rDNAs in DNA samples obtained not only from environments in which thermophilic archaea are abundant but also from environments in which methanogenic archaea are abundant. Our findings indicate that this method is applicable to culture-independent molecular analysis of microbial communities in various environments.

Archaea↗