Date of Graduation

5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Yanbin Li

Committee Member

Young Min Kwon

Second Committee Member

Mack Ivey

Third Committee Member

Jiangchao Zhao

Keywords

Whole-bacterium SELEX; Aptamer; E. coli O157:H7; QCM; RCA

Abstract

Escherichia coli O157:H7 is one of the top five pathogens contributing to foodborne diseases, causing an estimated 2,138 cases of hospitalization in the US each year. The extremely low infectious dose demands for more rapid and sensitive methods to detect E. coli O157:H7. The objective of this study is to select aptamers specifically binding to E. coli O157:H7 using whole-bacterium SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and to create a multivalent aptamer system by rolling circle amplification (RCA) with the selected aptamer sequence for sensitive detection of E. coli O157:H7 using a quartz crystal microbalance (QCM) sensor. Briefly, A total of 19 rounds of selection against live E. coli O157:H7 and 6 rounds of counter selection were performed for SELEX. One sequence S1 that appeared 16 (out of 20) times was characterized and a dissociation constant (Kd) of 10.30 nM was obtained. Using phi29 DNA polymerase, RCA reaction was performed, which produced a long ssDNA strand composed of thousands of repetitive aptamer sequences, termed as a multivalent aptamer system, on the electrode. The QCM sensor based on a multivalent aptamer system was able to quantitatively detect E. coli O157:H7. The limit of detection (LOD) of the QCM sensor was determined to be 34 CFU/ml, respectively, with the whole detection procedure in less than 40 min. The QCM sensor also showed high specificity for E. coli O157:H7 when it was cross-tested with five non-target bacteria. The QCM aptasensor in this study provided a common platform for detection of different foodborne pathogens.

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