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Title: Immunomagnetic separation and typing of a food-borne pathogen, Campylobacter jejuni
Author: Needham, Simon Andrew
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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PEB3, a cell envelope protein, was identified as a target for use in an immunomagnetic isolation system (IMS). Peb3 was cloned into Escherichia coli and expressed as a His-tagged construct (His.PEB3). Rabbits were then challenged with the purified construct to produce polyclonal antisera. Isolation of C. jejuni from a mixed culture (E. coli or Arcobacter spp. and C. jejuni) with polyclonal antisera was attempted but failed to capture whole C. jejuni cells. C. jejuni expresses two cell envelope associated polysaccharides: a short chain lipooligosaccharide (LOS) and a long chain lipid-linked polysaccharide, capsular polysaccharide (CPS). The Penner serotyping system has been shown to use CPS as its major discriminatory determinant. A novel molecular based typing system was devised based upon restriction fragments length polymorphism (RFLP) using the CPS locus as a discriminatory determinant. Primers for use in long template PCR (LT-PCR) were designed against the genes, kpsD, kpsS, gmhA2 and cj1430. The theoretical maximum amplicon length obtainable in LT-PCR is ~40 kb; however, this is dependent on a number of factors including the %GC of the target amplicon. The low %GC of C. jejuni DNA prevented the amplification of the entire locus length (~41 kb) in one reaction and by experimentation the maximum amplifiable length was determined to be 25 kb. This maximum length was only achieved sporadically and the maximum length that could be reliably amplified was ~15 kb. Because of this limitation only the amplicon kpsS-gmhA2 was used in the typing scheme. Amplification was only achieved with the type strain, C. jejuni NCTC 11168. This is believed to be due to difficulties in amplifying low %GC targets with the ExpandTM 20 kb plus long template system (Roche).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available