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Title: Nucleic acid approaches to toxin detection
Author: Chatwell, Nicola
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2013
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PCR is commonly used for detecting contamination of foods by toxigenic bacteria. However, it is unknown whether it is suitable for detecting toxins in samples which are unlikely to contain bacterial cells, such as purified biological weapons. Quantitative real-time PCR assays were developed for amplification of the genes encoding Clostridium botulinum neurotoxins A to F, Staphylococcal enteroxin B (SEB), ricin, and C. perfringens alpha toxin. Botulinum neurotoxins, alpha toxin, ricin and V antigen from Yersinia pestis were purified at Dstl using methods including precipitation, ion exchange, FPLC, affinity chromatography and gel filtration. Additionally, toxin samples of unknown purity were purchased from a commercial supplier. Q-PCR analysis showed that DNA was present in crudely prepared toxin samples. However, the majority of purified or commercially produced toxins were not detectable by PCR. Therefore, it is unlikely that PCR will serve as a primary toxin detection method in future. Immuno-PCR was investigated as an alternative, more direct method of toxin detection. Several iterations of the method were investigated, each using a different way of labelling the secondary antibody with DNA. It was discovered that the way in which antibodies are labelled with DNA is crucial to the success of the method, as the DNA concentration must be optimised in order to fully take advantage of signal amplification without causing excessive background noise. In general terms immuno-PCR was demonstrated to offer increased sensitivity over conventional ELISA, once fully optimised, making it particularly useful for biological weapons analysis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available