title: Bioengineering bacteriophages to enhance the sensitivity of phage amplification-based paper fluidic detection of bacteria.
abstract: Bacteriophage (phage) amplification is an attractive method for the detection of bacteria due to a narrow phage-host specificity, short amplification times, and the phages' ability to differentiate between viable and non-viable bacterial cells. The next step in phage-based bacteria detection is leveraging bioengineered phages to create low-cost, rapid, and easy-to-use detection platforms such as lateral flow assays. Our work establishes the proof-of-concept for the use of bioengineered T7 phage strains to increase the sensitivity of phage amplification-based lateral flow assays. We have demonstrated a greater than 10-fold increase in sensitivity using a phage-based protein reporter, maltose-binding protein, over the detection of replicated T7 phage viron itself, and a greater then 100-fold increase in sensitivity using a phage-based enzymatic reporter, alkaline phosphatase. This increase in sensitivity enabled us to detect 10(3)CFU/mL of Escherichia coli in broth after 7h, and by adding a filter concentration step, the ability to detect a regulatory relevant E. coli concentration of 100CFU/100mL in inoculated river water after 9h, where the current standard requires days for results. The combination of the paper fluidic format with phage-based detection provides a platform for the development of novel diagnostics that are sensitive, rapid, and easy to use.
authors.affiliation: Department of Food Science, University of Massachusetts, Amherst, MA, United States.
authors.forename: S D
authors.initials: SD
authors.lastname: Alcaine
authors.affiliation: Department of Microbiology, University of Massachusetts, Amherst, MA, United States.
authors.forename: K
authors.initials: K
authors.lastname: Law
authors.affiliation: Department of Microbiology, University of Massachusetts, Amherst, MA, United States.
authors.forename: S
authors.initials: S
authors.lastname: Ho
authors.affiliation: Department of Food Science, University of Massachusetts, Amherst, MA, United States.
authors.forename: A J
authors.initials: AJ
authors.lastname: Kinchla
authors.affiliation: Department of Food Science, University of Massachusetts, Amherst, MA, United States; Department of Microbiology, University of Massachusetts, Amherst, MA, United States.
authors.forename: D A
authors.initials: DA
authors.lastname: Sela
authors.affiliation: Department of Food Science, University of Massachusetts, Amherst, MA, United States; Department of Microbiology, University of Massachusetts, Amherst, MA, United States. Electronic address: snugen@umass.edu.
authors.forename: S R
authors.initials: SR
authors.lastname: Nugen
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics*; D057005:Bioengineering / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods*; D004784:Environmental Monitoring / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification* / Q000821:virology*; D005818:Genetic Engineering / Q000379:methods; D057230:Limit of Detection; D058619:Maltose-Binding Proteins / Q000235:genetics; D045483:Rivers / Q000382:microbiology*; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D058619:Maltose-Binding Proteins; D000469:Alkaline Phosphatase
keywords: Bacteriophage; Biosensor, water quality; E. coli; Genetic engineering; Lateral flow assay; Synthetic biology