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Reporter phage assays are a promising alternative to culture-based assays for rapidly detecting viable bacteria. The reporter systems used in phage-based detection are typically enzymes and their corresponding substrates that provide a signal following infection and expression. While several reporter systems have been developed, comparing reporter systems based on reported bacteria detection limits from literature can be challenging due to factors other than the reporter system that influence detection capabilities. To advance the development of phage-based assays, a systematic comparison and understanding of the components are necessary. The objective of this study was to directly compare two common enzyme-mediated luminescence reporter systems, NanoLuc/Nano-Glo and alkaline phosphatase (ALP*)/DynaLight, for phage-based detection of bacteria. The detection limits of the purified enzymes were determined, as well as the expression levels and bacteria detection capabilities following engineering of the coding genes into T7 phage and infection of E. coli BL21. When comparing the sensitivity of the purified enzymes, NLuc/Nano-Glo enzyme/substrate system demonstrated a lower detection limit than ALP*/DynaLight. In addition, the expression of the NLuc reporter following phage infection of E. coli was greater than ALP*. The lower detection limit combined with the higher expression resulted in a greater than 100-fold increase in sensitivity for the NLuc/Nano-Glo ® reporter system compared to ALP*/DynaLight when used for the detection of E. coli in a model system. These findings provide a comparative analysis of two common reporter systems used for phage-based detection of bacteria and a foundational understanding of these systems for engineering future reporter phage assays.

Comparative Analysis of NanoLuc Luciferase and Alkaline Phosphatase Luminescence Reporter Systems for Phage-Based Detection of Bacteria