Production of DNA aptamers with specificity for bacterial food pathogens

Hdl Handle:
http://hdl.handle.net/10034/620695
Title:
Production of DNA aptamers with specificity for bacterial food pathogens
Authors:
Kärkkäinen, Riikka M.
Abstract:
Aptamers are biomolecular ligands composed of nucleic acids. They can be selected to bind specifically to a range of target molecules and subsequently exploited in a fashion analogous to more traditional biomolecules such as antibodies. In this study a method for selecting new aptamers which specifically bind whole live bacterial cells is described. A non-pathogenic strain of Escherichia coli K12 was used to develop the method. A DNA library containing 100 bases long random nucleotide sequences was produced and the aptamer selection process was repeated nine times. An enzyme-linked technique was first used to detect bound aptamers thereafter fluorimetry and fluorescence microscopy methods were used for the detection. The aptamers were cloned and sequenced and the cloned aptamers produced with fluorescent labels. The E. coli K12-binding aptamers were used to demonstrate the detection of the bacterial cells in a complex food matrix, namely probiotic yogurt, by using fluorescence based detection method. The aptamer selection method with some modifications was also used to select aptamers with specificity for the food pathogens E. coli O157, Listeria monocytogenes, L. innocua, S. typhimurium and S. enteritidis. The aptamers against E. coli O157 and S. typhimurium were cloned and the sequences and the binding properties of these aptamers were analysed. The use of E. coli K12 as a target organism and the aptamer sequences presented in this study, have not previously been published in scientific literature. This is also the first report where the aptamers have been used in detection of live bacterial cells in yogurt.
Advisors:
Bonwick, Graham A.; Drasbek, Mette R.; Young, Niall; Smith, Christopher; McDowall, Ian
Citation:
Kärkkäinen, R. M. (2012). Production of DNA aptamers with specificity for bacterial food pathogens (Doctoral dissertation). University of Chester: United Kingdom.
Publisher:
University of Chester
Publication Date:
Sep-2012
URI:
http://hdl.handle.net/10034/620695
Type:
Thesis or dissertation
Language:
en
Appears in Collections:
Theses

Full metadata record

DC FieldValue Language
dc.contributor.advisorBonwick, Graham A.en
dc.contributor.advisorDrasbek, Mette R.en
dc.contributor.advisorYoung, Niallen
dc.contributor.advisorSmith, Christopheren
dc.contributor.advisorMcDowall, Ianen
dc.contributor.authorKärkkäinen, Riikka M.en
dc.date.accessioned2017-10-30T15:23:06Z-
dc.date.available2017-10-30T15:23:06Z-
dc.date.issued2012-09-
dc.identifier.citationKärkkäinen, R. M. (2012). Production of DNA aptamers with specificity for bacterial food pathogens (Doctoral dissertation). University of Chester: United Kingdom.en
dc.identifier.urihttp://hdl.handle.net/10034/620695-
dc.description.abstractAptamers are biomolecular ligands composed of nucleic acids. They can be selected to bind specifically to a range of target molecules and subsequently exploited in a fashion analogous to more traditional biomolecules such as antibodies. In this study a method for selecting new aptamers which specifically bind whole live bacterial cells is described. A non-pathogenic strain of Escherichia coli K12 was used to develop the method. A DNA library containing 100 bases long random nucleotide sequences was produced and the aptamer selection process was repeated nine times. An enzyme-linked technique was first used to detect bound aptamers thereafter fluorimetry and fluorescence microscopy methods were used for the detection. The aptamers were cloned and sequenced and the cloned aptamers produced with fluorescent labels. The E. coli K12-binding aptamers were used to demonstrate the detection of the bacterial cells in a complex food matrix, namely probiotic yogurt, by using fluorescence based detection method. The aptamer selection method with some modifications was also used to select aptamers with specificity for the food pathogens E. coli O157, Listeria monocytogenes, L. innocua, S. typhimurium and S. enteritidis. The aptamers against E. coli O157 and S. typhimurium were cloned and the sequences and the binding properties of these aptamers were analysed. The use of E. coli K12 as a target organism and the aptamer sequences presented in this study, have not previously been published in scientific literature. This is also the first report where the aptamers have been used in detection of live bacterial cells in yogurt.en
dc.language.isoenen
dc.publisherUniversity of Chesteren
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/*
dc.subjectDNAen
dc.subjectfood pathogensen
dc.subjectpathogen detectionen
dc.subjectSELEXen
dc.titleProduction of DNA aptamers with specificity for bacterial food pathogensen
dc.typeThesis or dissertationen
dc.rights.embargodate2017-10-05-
dc.type.qualificationnamePhDen
dc.type.qualificationlevelDoctoralen
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