Evaluation of NanoLuc and GFP Reporter-labeled Control Strains for Shiga Toxin-producing Escherichia coli (STEC) Assays

Positive controls are essential for establishing assay performance and equipment efficacy. Yet, some food testing laboratories refrain from using bacterial strains as positive controls for fear of cross-contaminating their samples. Under the Food and Drug Administration (FDA) Food Safety Modernization Act, laboratories face an increasing number of regulations to expand testing for objectionable organisms. Control strains with unique, easily detectable traits which distinguish positive control strains from actual food contaminants can help differentiate true contamination from control strain cross-contamination.

In this study, we introduced shuttle vectors encoding either green fluorescent protein (GFP, Life Technologies) or NanoLuc (Promega) into Escherichia coli strains, including Shiga toxin-producing O157 (stx1⁺, stx2⁺, eaeA⁺) and non-Shiga toxin-producing O157 (stx1^-, stx2^-, eaeA^-), for use in food pathogen detection assays. Both reporters can be easily visualized without specialized detection equipment; GFP fluoresces when excited by UV light, while bacteria engineered with NanoLuc emit a strong light signal in the presence of a chemical substrate. Upon establishing the detectability of NanoLuc in the E. coli O157 strains, the reporter was transformed into the “Big Six” non-O157 E. coli strains (serogroups: O26, O45, O103, O111, O121, and O145) for use as reporter-labeled positive controls.