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Title: Strain differentiation of Yersinia pestis
Author: Blackman, Lois
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2001
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Verification procedures are required to ensure compliance with the Biological and Toxins Weapons Convention (BTWC). Strain differentiation analysis may need to be performed on samples that do not harbour live bacteria and may contain other microorganisms. Thus the method employed must be non-culture based and specific to the bacterial species. Three Polymerase Chain Reaction (PCR) based assays were evaluated for their potential to specifically differentiate strains of Yersinia pestis. Two of the techniques, Insertion Sequence - Flanking Region Amplification PCR (IS-FRAP) and Variable Number of Tandem Repeats - PCR (VNTR-PCR), demonstrated specific and reproducible differentiation of a test panel of 84 Y. pestis isolates. These isolates included biotyped and/or ribotyped strains, previously untyped isolates and repetitive subcultures of Y. pestis CO92. Sites of chromosomal and plasmid insertion of the IS100 element were analysed by IS-FRAP. The amplification profiles divided the test panel isolates into 16 types. Two octonucleotide VNTR sequences were identified in the proximity of the same IS100 element. The observation of 11 alleles of VNTR-8A upstream of 4 alleles of VNTR-8B enabled the differentiation of the test panel isolates into 19 VNTR-PCR types. Strains isolated from similar geographical locations often displayed the same VNTR-PCR type. These PCR techniques were developed into prototype assays that may be suitable for the analysis of BTWC samples. Homologous sequences to the Y. pestis VNTR regions were analysed in Y. pseudotuberculosis strains. The octonucleotide sequence of VNTR-8A was not repeated in Y. pseudotuberculosis and was flanked by 361bp of DNA not identified in Y. pestis strains. Recombination events leading to the formation of the VNTR are hypothesised.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available