Flow‐Cytometry‐Based Method to Detect Escherichia coli and Shigella Spp. Using 16S rRNA‐Based Probe

Yong Xue1, Jon G. Wilkes1, Ted J. Moskal2, Anna J. Williams1, Willie M. Cooper1, Dan A. Buzatu1

1 Division of Systems Biology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas, 2 Life Sciences Consultant, Jonesboro, Arkansas
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.25
DOI:  10.1002/cptx.14
Online Posting Date:  February, 2017
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Abstract

Detection of microbial contamination in foods before they go on to the market can help prevent the occurrence of foodborne illness outbreaks. Current methods for the detection of Escherichia coli are limited by time‐consuming procedures, which include multiple culture incubation steps, and require several days to get results. This unit describes the development of an improved rapid flow‐cytometry‐based detection method that has greater sensitivity and specificity. This method requires less time‐to‐results (TTR) and can detect a small number of E. coli in the presence of large numbers of other bacteria. Clear step‐by‐step protocols for cell concentration determination, sample preparation, and flow cytometric analysis are provided. © 2017 by John Wiley & Sons, Inc.

Keywords: flow cytometry; foodborne illness; Escherichia coli; microbial contamination; oligonucleotide probe; Shigella; Salmonella

     
 
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Table of Contents

  • Basic Protocol 1:  
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:  

  Materials
  • Brain heart infusion (BHI) medium
  • Nutrient broth (NB) medium
  • Trypticase soy broth (TSB) medium
  • A generic E. coli oligonucleotide probe ES445 (Kenzaka et al., ) with sequence: 5′ CTT TAC TCC CTT CCT CCC 3′ with Alexa Fluor 488 linked to its 5′ end
  • 1× PBS solution
  • 10% buffered formalin
  • 50% ethanol/PBS solution
  • Hybridization buffer (20 mM Tris, pH 7.5, 0.01% SDS, 3 M NaCl; see recipe)
  • Total plate count (TPC) reagent containing thiazole orange (TO) and propidium iodide (PI)
  • 37°C incubator
  • Benchtop centrifuge
  • Thermomixer B
  • Flow cytometer
  • Data analysis software: FCS Express 4 Flow Cytometry software
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Figures

Videos

Literature Cited

Literature Cited
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  Centers for Disease Control and Prevention. 2011. Reports of Selected E. coli Outbreak Investigations. http://www.cdc.gov/ecoli/outbreaks.html (Accessed July 6, 2016).
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  Xue, Y., Wilkes, J.G., Moskal, T.J., Williams, A.J., Cooper, W.M., Nayak, R., Rafii, F., and Buzatu, D.A. 2016. Development of a flow cytometry‐based method for rapid detection of Escherichia coli and Shigella Spp. using an oligonucleotide probe. PLoS One. 11:e0150038. doi: 10.1371/journal.pone.0150038.
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