published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Microbiology / Master / Master of Medical Sciences

No description available.

No description available.

Amplification techniques such as the Polymerase Chain Reaction (PCR) are held to be largely qualitative procedures and are widely used as such. Since the efficiency of amplification is less than perfect, small changes in efficiency can yield dramatic differences in the final amount of product generated. Despite this unpredictability the exquisite sensitivity of PCR makes the demanding goal of absolute quantification highly desirable. Consequently, the use of this technique for the quantification of nucleic acids has increased at an exponential rate. However, the ability of PCR to accurately quantify absolute levels of DNA is still not universally accepted. The overall aim of this investigation was to determine the critical factors affecting the quantification of DNA using PCR and to use these findings to develop an assay for the absolute quantification of DNA in a model system. The novel work presented here illustrates the need for careful examination of sequencesfo r GC-rich domains which could give rise to stable secondary structures and reduce the efficiency of amplification by serving as termination sites. To determine the accuracy of competitive PCR, CE and IP-RP-HPLC were employed to quantify PCR- products. These two techniques provided valuable information on the identification and elimination of sources of error which led to improvements in speed, accuracy and precision, as well as ease of quantification by PCR. They also yielded information on the process of heteroduplex formation whilst simultaneously revealing assay limitations. Consequently, the on-line fluorescence monitoring of PCR was used as an alternative method for the quantification of Legionella pneumophila. This technique was highly reproducible however, mispriming and the subsequent amplification of non-specific PCR products limited the level of detection. The Y-end labelling of degraded DNA with DIG prevented short DNA fragments from mispriming (and consequently extending) allowing the amplification of DNA targets. Therefore, to reduce mispriming and hence improve assay sensitivity, this approach was adapted for the first time to produce 5'-degenerate, 3'- DIG-terminated competitive primer analogues. These analogues, coupled with the use of the LightcyclerTm, allowed the detection and absolute quantification of a single cell of Legionella pneumophila. This is the first time that this level of sensitivity has been achieved using this type of assay. This technique should provide a very rapid and sensitive alternative for quantification comparedt o the other,m oree xpensivete chnologiesa vailablea t present.

This work has compared polymerase chain reaction (PCR) assays and traditional visual disease assessment to evaluate the severity of Fusarium ear blight (FEB) ofwheat under field and glasshouse conditions. In a field trial, PCR analysis highlighted the problem of diagnosis of FEB of wheat based on visual disease assessment where natural inoculum was present. PCR-based analysis detected F. poae predominantly in the glumes and Microdochium nivale sub-species were predominantly found in the rachis component of ears. M nivale var. majus was more frequently observed than var. nivale (64 and 36 %, respectively). Quantitative PCR analysis and conventional visual disease assessment were used to evaluate fungicide efficacy against FEB of wheat caused by F. culmorum and F. poae in three glasshouse trials (1994/5-1996/7). Prochloraz and tebuconazole significantly decreased both visual symptoms of FEB and fungal DNA content of F. culmorum and F. poae ear blight of wheat. Overall, both fungicides appeared equally effective, although the efficacy ofthese fungicides was consistently greater as measured by PCR analysis rather than by visual disease assessment. Inoculation with F. culmorum significantly reduced yield (1000 grain weight) whereas inoculation with F. poae had no significant effect on yield. Fusarium culmorum was successfully transformed with the GUS reporter gene. GUS activity levels of transformants varied, but transformation did not affect pathogenicity on wheat seedlings. A GUS transformant was used to study the effectiveness of two fungicides against F. culmorum foot rot of wheat. Primers to the Tri5 gene were used to develop a PCR-based assay for the specific detection ofpotential trichothecene-producing Fusarium species. These primers were also used to develop an RT-PCR-based assay for the detection and semiquantification of F. culmorum Tri5 gene expression 'relative to p-tubulin gene expression' in RNA extracts from F. culmorum. This assay was used to show that time and fungicides can affect Tri5 gene expression in liquid culture.

Thesis (Ph.D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (April 25, 2007) Vita. Includes bibliographical references.

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 40-46).

Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. Jianlin Cheng. Includes bibliographical references.

Thesis (Ph. D.)--University of Queensland, 2003. / Includes bibliographical references.