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Title: The detection of bacterial spores
Author: Hindle, A.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1999
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A rapid analytical method capable of detecting low concentrations of bacterial spores has been developed. Dipicolinic acid (DPA), a universal and specific component of bacterial spores, was chosen as a suitable target analyte for bacterial spore detection. DPA was assayed by fluorimetric detection of its chelates with lanthanide metals. The influence of the choice and concentration of lanthanide and buffer ions on the fluorescence assay was studied as well as the effects of pH and temperature. The optimal system had a detection limit of 2nM DPA. This assay allowed the first real-time monitoring of the germination of bacterial spores by continuously quantifying exuded DPA. A detection limit of 104 B. subtilis spores/ml was reached, representing a substantial improvement over previous rapid tests. To further improve the detection limit, a selective preconcentration step was investigated; both biological and chemical recognition molecules were compared for this purpose. A novel DPA analogue was synthesized and immobilized which was more suitable for use as an antigen than previous DPA conjugates. Several conjugates were then applied to screening a phage-antibody library for anti-DPA recognition molecules; however, the library was found not to contain appropriate phage-antibodies. Other means of concentrating DPA were considered and successful preconcentration was demonstrated with Dowex-1 anion-exchange resin. DPA was concentrated from a large volume of solution then eluted directly into the optimum fluorescence assay mixture allowing a convenient analytical method. A 66-fold concentration of DPA was demonstrated, resulting in a limit of detection of 50pM DPA or 133 spores/ml. from a 1L sample of aqueous suspension. This is the first rapid analytical method for bacterial spores which is sufficiently sensitive for industrial application. Future prospects for bacterial spore detection and further improvements to both the fluorescence assay and the preconcentration step were discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available