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Document Type

Campus-Only Access for Five (5) Years

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Food Science

Year Degree Awarded

2016

Month Degree Awarded

September

First Advisor

Dr. Sam R. Nugen

Second Advisor

Dr. David A. Sela

Third Advisor

Dr. Steven J. Sandler

Subject Categories

Food Science

Abstract

Foodborne or waterborne pathogens which are responsible for numerous worldwide outbreaks of disease have caused serious health problems and enormous economic cost. Development of new bacterial detection technologies with high sensitivity and specificity is of great importance for food safety and public health. This has highlighted the significance of developing rapid and effective techniques for pathogen screening. Thus we aim to explore a bacteriophage mediated detection system for foodborne or waterborne bacteria monitoring.

Bacteriophage as a novel recognition probe has been applied in various biosensor by immobilizing phage particles on solid sensor surface. They possess high specificity for targeted microorganism and a rapid infection between phage and its host cell. Their characteristics including low cost, robustness, fast and easy production plus high tolerance under extreme conditions make phages superior for bacteria detection at resource-limited settings.

We utilize biotin-streptavidin interaction system for an engineered phage immobilization on biosensor surface. The fusion of biotin acceptor peptide (BAP) gene and biotin ligase (BirA) gene to phage capsid protein gene enabled the display of BAP ligand and the expression of protein BirA during the replication cycle of phage infection. Then produced phage progenies could be biotinylated in vivo and immobilized on the streptavidin coated magnetic beads. Compared with wild type phage, the recombinant phage showed a significantly higher immobilization efficiency of 82.8% on the magnetic bead and the resulting bio-probe showed a capture efficiency of 86.2% of E.coli within 20 min. This phage based biomagnetic separation coupled PCR detection provided a detection limit of 102 CFU/mL bacteria without additional pre-enrichment. For the detection of pathogenic Escherichia coli O157:H7, we compared phage-based biosorbent and antibody-based one and found that the capture efficiency of our phage assay was about 20% higher than that of antibody based magnetic separation assay under extreme conditions. Then we developed a membrane filter based colorimetric detection assay to offer a convenient and rapid way for the identification of general E.coli from field water on-site. This phage mediated detection provided a convenient and quantitative detection of bacteria number as the colored signal present at the site corresponding to the located bacterial colony.

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