Quantitative bioluminescence assay for measuring Bacillus cereus nonhemolytic enterotoxin complex

PLoS One. 2020 Sep 30;15(9):e0238153. doi: 10.1371/journal.pone.0238153. eCollection 2020.

Abstract

Bacillus cereus is a foodborne pathogen causing emesis and diarrhea in those affected. It is assumed that the non-hemolytic enterotoxin (Nhe) plays a key role in B. cereus induced diarrhea. The ability to trace Nhe activity is important for food safety. While assays such as PCR and ELISA exist to detect Nhe, those methods cannot differentiate between active and inactive forms of Nhe. The existing rabbit ileal loop bioassay used to detect Nhe activity is ethically disfavored because it uses live experimental animals. Here we present a custom built low-cost CCD based luminometer and applied it in conjunction with a cell-based assay using Vero cells transduced to express the luciferase enzyme. The activity of Nhe was measured as its ability to inhibit synthesis of luciferase as quantified by reduction of light emission by the luciferase reaction. Emitted light intensity was observed to be inversely proportional to Nhe concentration over a range of 7 ng/ml to 125 ng/ml, with a limit of detection of 7 ng/ml Nhe.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biocatalysis
  • Chlorocebus aethiops
  • Enterotoxins / metabolism*
  • HEK293 Cells
  • Humans
  • Luciferases / genetics
  • Luciferases / metabolism
  • Luminescent Measurements*
  • Vero Cells

Substances

  • Enterotoxins
  • enterotoxin, Bacillus cereus
  • Luciferases

Grants and funding

This project was supported by the United States Department of Agriculture, Agricultural Research Service (ARS). The authors are all employed by the ARS. There was no additional source of external funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.