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Production of DNA aptamers with specificity for bacterial food pathogens
Kärkkäinen, Riikka M.
Kärkkäinen, Riikka M.
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2012-09
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Doctoral thesis
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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.
Citation
Kärkkäinen, R. M. (2012). Production of DNA aptamers with specificity for bacterial food pathogens (Doctoral dissertation). University of Chester: United Kingdom.
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University of Chester
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Thesis or dissertation
Language
en