Absolute quantification of target DNA: a simple competitive PCR for efficient analysis of multiple samples

Abstract Using dissociation and enhancement time-resolved lanthanide fluorometry, we have developed a quantitative competitive (QC)-PCR for measuring parathyroid hormone-related protein (PTHrP) mRNA after reverse transcription. A cloned PTHrP cDNA target was also modified by deletion of 10 bp and insertion of 21 bp in the midregion of the fragment and cloned for use as a competitor (i.e., internal standard). Two primers spanning 362 bp of target and 373 bp of competitor were designed and one of the primers was biotinylated. Two oligonucleotide probes, one recognizing the target and the other hybridizing to the competitor, were labeled with Eu chelate. Two equal aliquots of PCR products were assayed with each probe separately in streptavidin-coated wells. After 35 PCR cycles, the competitor signal decreased exponentially (y = e(3.74 −0.624x); r2 = 0.965) and the target signal increased exponentially (y = e(1.14 + 0.497x); r2 = 0.984) when 1000 copies/tube of the competitor and 0–100 000 copies/tube of the target DNA were added. Log-transformed data for the ratio of target to competitor signals (y) and the copies of the target DNA added (x) were used for plotting the linear calibration curve (y = 2.79+2.76x; r2 = 0.976). This QC-PCR enables analysis of multiple samples simultaneously and can be used to study PTHrP gene expression in malignancy and physiology.

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Comparison of the ABI 7700 System (TaqMan) and Competitive PCR for Quantification of IS6110 DNA in Sputum during Treatment of Tuberculosis

Journal of Clinical Microbiology ◽

10.1128/jcm.36.7.1964-1968.1998 ◽

1998 ◽

Vol 36 (7) ◽

pp. 1964-1968 ◽

Cited By ~ 109

Author(s):

L. e. Desjardin ◽

Y. Chen ◽

M. D. Perkins ◽

L. Teixeira ◽

M. D. Cave ◽

...

Keyword(s):

Detection System ◽

Good Method ◽

Automated System ◽

Competitive Pcr ◽

Sequence Detection ◽

Antituberculosis Therapy ◽

Fluorogenic Probe ◽

Target Dna ◽

Monitoring Treatment ◽

Antituberculosis Chemotherapy

Mycobacterium tuberculosis can persist in sputum for long periods of time after the initiation of antituberculosis chemotherapy. The purpose of this study was to determine whether quantitative estimates of M. tuberculosis DNA in sputum correlate with the numbers of viable bacilli and thus measure the therapeutic response of patients during treatment. Two methods of M. tuberculosis DNA quantification were examined by using DNA isolated from sputum specimens serially collected during the course of chemotherapy. A competitive PCR assay was compared to an automated system of real-time quantification with the ABI Prism 7700 Sequence Detection System (TaqMan). The ABI 7700 system uses standard PCR in conjunction with a fluorogenic probe in which the intensity of fluorescence is proportional to the amount of target DNA present. The results showed that both PCR systems are reproducible and accurate. The amounts of M. tuberculosis DNA quantified in sputum corresponded well with the numbers of acid-fast bacilli (AFB) counted by microscopy. Before initiation of antituberculosis therapy, measures of AFB, M. tuberculosis DNA, and cultivable bacilli were similar, suggesting that quantification of DNA is a good method for measuring the initial bacillary load. However, the rate of disappearance of both AFB and M. tuberculosis DNA did not correlate with the decline in cultivable bacilli in the specimen; therefore, these tests are not appropriate for monitoring treatment efficacy.

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Absolute quantification of genetically engineered traits with droplet digital PCR: Effect of DNA treatments and spiking with non-target DNA

Food Control ◽

10.1016/j.foodcont.2016.03.007 ◽

2016 ◽

Vol 68 ◽

pp. 105-111 ◽

Cited By ~ 5

Author(s):

Tigst Demeke ◽

Michelle Holigroski ◽

Monika Eng ◽

Juan Xing

Keyword(s):

Digital Pcr ◽

Absolute Quantification ◽

Genetically Engineered ◽

Droplet Digital Pcr ◽

Target Dna

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Combination of Competitive Quantitative PCR and Constant-Denaturant Capillary Electrophoresis for High-Resolution Detection and Enumeration of Microbial Cells

Applied and Environmental Microbiology ◽

10.1128/aem.67.9.3897-3903.2001 ◽

2001 ◽

Vol 67 (9) ◽

pp. 3897-3903 ◽

Cited By ~ 16

Author(s):

Eelin L. Lim ◽

Aoy V. Tomita ◽

William G. Thilly ◽

Martin F. Polz

Keyword(s):

Capillary Electrophoresis ◽

High Resolution ◽

Quantitative Pcr ◽

Environmental Samples ◽

Model Organism ◽

Amplification Efficiency ◽

Competitive Pcr ◽

Microbial Cells ◽

Novel Approach ◽

Target Dna

ABSTRACT A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 104 cells ml−1. The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells ml−1 by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples.

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Absolute quantification of the budding yeast transcriptome by means of competitive PCR between genomic and complementary DNAs

BMC Genomics ◽

10.1186/1471-2164-9-574 ◽

2008 ◽

Vol 9 (1) ◽

pp. 574 ◽

Cited By ~ 64

Author(s):

Fumihito Miura ◽

Noriko Kawaguchi ◽

Mikio Yoshida ◽

Chihiro Uematsu ◽

Keiji Kito ◽

...

Keyword(s):

Budding Yeast ◽

Absolute Quantification ◽

Competitive Pcr

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Design and construction of a micromilled fluidic device as part of a DNA biosensor

Proceedings of the Institution of Mechanical Engineers Part C Journal of Mechanical Engineering Science ◽

10.1243/09544062jmes715 ◽

2008 ◽

Vol 222 (5) ◽

pp. 847-853 ◽

Cited By ~ 2

Author(s):

R J Townsend ◽

N R Harris ◽

D Wenn ◽

D Brennan ◽

N J Grabham

Keyword(s):

Dna Sequences ◽

Single Sample ◽

Fluid Sample ◽

Single Chip ◽

Novel Approach ◽

Design Changes ◽

Target Dna ◽

Fluidic Device ◽

Parallel Tests ◽

Multiple Samples

Under the Optonanogen project (EU contract IST-2001-37239), a novel biosensor has been developed, which incorporates a disposable acrylic polymethylmethacrylate (PMMA) fluidic header. This header is designed to deliver a sample to a series of chemically primed cantilevers where hybridization of target DNA sequences and resulting deflection of the cantilevers is detected optically. Two different microfluidic headers are described, which are designed to incorporate the cantilever chip and which demonstrate a novel approach to microfluidic header assembly, integration with macroscale fluidics, fluidic handling, and priming strategies. The first header facilitates the delivery of a single fluid sample to all cantilevers, whereas the second permits discrete delivery of samples to isolated cantilevers, despite all cantilevers being contained on a single chip. This second, multi-path header therefore allows simultaneous analysis of multiple samples, or multiple parallel tests on a single sample. This paper describes these headers and for the multi-path device details the design changes incorporated to ensure effective isolation of the sample including a novel valve to improve priming of the microfluidic circuit.

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Method for absolute quantification of microbial communities by using both microarrays and competitive PCR

Journal of Microbiological Methods ◽

10.1016/j.mimet.2019.105718 ◽

2019 ◽

Vol 165 ◽

pp. 105718 ◽

Cited By ~ 1

Author(s):

Ayaka Yazawa ◽

Shigeki Kamitani ◽

Naoyuki Togawa

Keyword(s):

Microbial Communities ◽

Absolute Quantification ◽

Competitive Pcr

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Short Stress State Questionnaire

European Journal of Psychological Assessment ◽

10.1027/1015-5759/a000200 ◽

2015 ◽

Vol 31 (1) ◽

pp. 20-30 ◽

Cited By ~ 28

Author(s):

William S. Helton ◽

Katharina Näswall

Keyword(s):

Stress State ◽

Factor Structure ◽

Human Performance ◽

Self Report ◽

Confirmatory Factor ◽

Stress States ◽

Related Stress ◽

Using Data ◽

Task Conditions ◽

Multiple Samples

Conscious appraisals of stress, or stress states, are an important aspect of human performance. This article presents evidence supporting the validity and measurement characteristics of a short multidimensional self-report measure of stress state, the Short Stress State Questionnaire (SSSQ; Helton, 2004 ). The SSSQ measures task engagement, distress, and worry. A confirmatory factor analysis of the SSSQ using data pooled from multiple samples suggests the SSSQ does have a three factor structure and post-task changes are not due to changes in factor structure, but to mean level changes (state changes). In addition, the SSSQ demonstrates sensitivity to task stressors in line with hypotheses. Different task conditions elicited unique patterns of stress state on the three factors of the SSSQ in line with prior predictions. The 24-item SSSQ is a valid measure of stress state which may be useful to researchers interested in conscious appraisals of task-related stress.

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