Caractérisation et détection par des méthodes génotypiques des agents bactériens aéroportés associés au bioterrorisme

Authors: Isabel, Sandra
Advisor: Bergeron, Michel G.Boissinot, Maurice
Abstract: This doctoral thesis presents four studies regarding the detection of airborne bacterial biothreat agents. First of all, a historical review of biological warfare and bioterrorism will allow for better understanding of this threat. To focus on the current problematic, biological agents, intervention procedures of public security and health organisations, as well as detection methods will then be introduced. The lack of systems for the reliable and rapid detection of biological agents is an important limitation of intervention procedures. Many interrelated steps are required to detect and identify biological agents. They consist of sampling, sample matrix processing, and finally, microbial identification by decoding macromolecule signatures, notably using genotyping methods. Some particles potentially employed to produce airborne biological weapons could interfere with molecular detection methods. Moreover, inoffensive powders (e.g. flour) can be used as hoaxes to terrorise individuals, organisations, or the population. Therefore, the first article studied the interference on PCR detection of 23 powdery or other environmental samples. This study presents a method to separate Bacillus spores from powdery matrices to alleviate the impact of these interfering compounds. The developed procedure is fast (≤10 minutes), inexpensive (~ 10$), allows easy handling (10 steps) and treatment of a wider sample variety compared to prior art. Secondly, a bacterial spore lysis based on rapid sonication (45 seconds) allowed extraction of genomic DNA in yields comparable to a robust commercialised lysis procedure requiring 5 minutes. The Fluidic Ultrasonic Lysis Module can be decontaminated and transferred from a contaminated to a secured area, which is advantageous in the context of bioterrorism. The third project shows the development of a detection assay based on a bacterial broad-spectrum PCR amplification of the target gene tuf and the identification of signature DNA sequences using a microfluidic microarray system. This assay allowed the rapid (75 minutes) detection of 12 of the 13 airborne bacterial biothreat agents listed by the CDC. Finally, analyses of tuf gene sequences from the Yersinia genus showed a high level of divergence between intra-genomic tufA and tufB sequences (8.3 to 16.2 %). This unexpected result could reveal particular circumstances allowing duplicated genes to acquire new functions. Moreover, this study allowed a phylogenetic analysis of 14 of the 17 described Yersinia species and added information about their taxonomical classification. In the near future, it would be interesting to combine the different biothreat detection steps presented here into a total analysis system. Hence, automation could facilitate its utilisation in the field to detect and identify (~1 h) biothreat agents resulting in more efficient medical management of victims and contribute to stop propagation.
Document Type: Thèse de doctorat
Issue Date: 2013
Open Access Date: 19 April 2018
Permalink: http://hdl.handle.net/20.500.11794/24517
Grantor: Université Laval
Collection:Thèses et mémoires

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