Skip to main content
Article
Reporter bacteriophage T7NLC utilizes a novel NanoLuc::CBM fusion for the ultrasensitive detection of Escherichia coli in water.
The Analyst
  • T.C. Hinkley, Cornell University
  • S. Garing, Intellectual Ventures Laboratory/Global Good
  • S. Singh, Iowa State University
  • A-L. M. Le Ny, Intellectual Ventures Laboratory/Global Good
  • K.P. Nichols, Iowa State University
  • J.E. Peters, Cornell University
  • Joey N. Talbert, Iowa State University
  • S.R. Nugen, Cornell University
Document Type
Article
Publication Version
Published Version
Publication Date
8-2-2018
DOI
10.1039/C8AN00781K
Abstract

Rapid detection of bacteria responsible for foodborne diseases is a growing necessity for public health. Reporter bacteriophages (phages) are robust biorecognition elements uniquely suited for the rapid and sensitive detection of bacterial species. The advantages of phages include their host specificity, ability to distinguish viable and non-viable cells, low cost, and ease of genetic engineering. Upon infection with reporter phages, target bacteria express reporter enzymes encoded within the phage genome. In this study, the T7 coliphage was genetically engineered to express the newly developed luceriferase, NanoLuc (NLuc), as an indicator of bacterial contamination. While several genetic approaches were employed to optimize reporter enzyme expression, the novel achievement of this work was the successful fusion of the NanoLuc reporter to a carbohydrate binding module (CBM) with specificity to crystalline cellulose. This novel chimeric reporter (nluc::cbm) bestows the specific and irreversible immobilization of NanoLuc onto a low-cost, widely available crystalline cellulosic substrate. We have shown the possibility of detecting the immobilized fusion protein in a filter plate which resulted from a single CFU of E. coli. We then demonstrated that microcrystalline cellulose can be used to concentrate the fusion reporter from 100 mL water samples allowing a limit of detection of <10 CFU mL-1 E. coli in 3 hours. Therefore, we conclude that our phage-based detection assay displays significant aptitude as a proof-of-concept drinking water diagnostic assay for the low-cost, rapid and sensitive detection of E. coli. Additional improvements in the capture efficiency of the phage-based fusion reporter should allow a limit of detection of <10 CFU per 100 mL.

Comments

This article is published as T.C. Hinkley, S. Garing, S. Singh, A-L.M. Le Ny, K.P. Nichols, J.E. Peters, J.N.Talbert, S.R. Nugen, Reporter bacteriophage T7NLC utilizes a novel NanoLuc::CBM fusion for the ultrasensitive detection of Escherichia coli in water. The Analyst; 2018. DOI: 10.1039/C8AN00781K.

Creative Commons License
Creative Commons Attribution 3.0
Copyright Owner
Royal Society of Chemistry
Language
en
File Format
application/pdf
Citation Information
T.C. Hinkley, S. Garing, S. Singh, A-L. M. Le Ny, et al.. "Reporter bacteriophage T7NLC utilizes a novel NanoLuc::CBM fusion for the ultrasensitive detection of Escherichia coli in water." The Analyst (2018)
Available at: http://works.bepress.com/joey_talbert/4/