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Theses and Dissertations

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Browsing Theses and Dissertations by Subject "accuracy"

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    Application of large-scale particle image velocimetry at the Hydraulics Laboratory of Colorado State University
    (Colorado State University. Libraries, 2018) Chen, Kaiwei, author; Ettema, Robert, advisor; Thornton, Christopher, committee member; Nelson, Peter, committee member; Landers, Stuart, committee member
    Large-scale particle image velocimetry (LSPIV) is a nonintrusive technique used to measure free-surface velocities of water flow in a manner that produces a two-dimensional vector field of flow velocity. LSPIV is gradually becoming quite widely used as a technique for measuring flow velocities in a range of flow areas. This study used readily available material and devices, and software, to apply LSPIV to flow fields in two laboratory flumes at the Hydraulics Laboratory of Colorado State University; LSPIV had not been used in this laboratory before this study. The applications used pieces of paper as tracer floats in the flow field, and employed a standard iPhone 6s to record video of the tracers moving in the flow field. The video record of tracer movements was then analyzed using Fudaa LSPIV software and Tecplot 360 software to calculate and present the flow velocity data. The applications demonstrated the utility of the LSPIV technique for determining the free-surface flow patterns, and their variations, in experiments done at the Hydraulics Laboratory. Additionally, this study examined the relationship between the tracer size and LSPIV accuracy with the objective of identifying an optimal width of tracer relative to the width of the flow field and its features. Five sizes of tracer were used in measuring the water-surface flow field through a series of contractions and expansions. It was found that the best tracer size is about from 3.80% to 6.33% of the wide of the channel.
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    ItemOpen Access
    Determining the effect of primer mismatches on quantitative PCR accuracy and developing guidance for design of primers with sequence variations
    (Colorado State University. Libraries, 2012) Ledeker, Brett Michael, author; De Long, Susan K., advisor; Omur-Ozbek, Pinar, committee member; Reardon, Kenneth F., committee member
    Although quantitative PCR (qPCR) is a powerful tool for investigating environmental systems, target gene sequences for organisms of interest often are not well known, which has resulted in few reliable primers for many applications. Additionally, the sequences of target genes found in diverse strains often contain sequence variations, and therefore, primer sets containing single or multiple primer-template mismatches are common. However, the detrimental impact of these mismatches on quantification accuracy and amplification efficiency has not been investigated thoroughly. Thus, the research objectives of this study were to elucidate the relationships between primer mismatches and the accuracy of qPCR assays and to develop guidance for designing primers targeting genes displaying sequence variations. The pcrA gene (encoding perchlorate reductase) from Dechloromonas agitata was used as a model system for this study, and a linearized plasmid containing the cloned pcrA gene was used as the qPCR template. A large number of pcrA primers (16 forward and 16 reverse) were designed containing from zero to three mismatches at various locations. Combinations of primers were tested to determine the impact of mismatches on the amplification efficiency, the threshold cycle (CT), and the quantification accuracy. Quantification accuracy was calculated as the percent detected by dividing the quantity measured with mismatch primers by the quantity measured with perfect match primers and multiplying by 100. Single mismatches at the 3' end resulted in quantification accuracies as low as ~3%, and single mismatches at the 5' end resulted in quantification accuracies as low as ~33%. Double and triple mismatches at the 5' resulted in quantification accuracies as low as ~17% and ~2%, respectively. Reductions in quantification accuracy correlated with increases in CT induced by mismatches but not with changes in amplification efficiency. Combining mismatched forward and reverse primers had an impact equivalent to the combined effect of the individual mismatch primers. Analogous qPCR tests were run with three other model genes: celS (encoding family 48 cellulase), C23O (encoding catechol dioxygenase, involved in toluene degradation), and hydA (encoding periplasmic hydrogenase, involved in fermentation). Primers were artificially designed to contain mismatches with these target genes, and results demonstrated that single or double mismatches can have a substantial detrimental impact on quantification accuracy in a broad range of systems. The results of this study indicate that caution must be taken to avoid mismatches when designing qPCR primers targeting genes with sequence variations and the findings serve to guide future design of primers for accurately quantifying genes in environmentally relevant systems.