Sensitive multiplex detection of whole bacteria using self-assembled cell binding domain complexes

Detecting bacterial cells at low levels is critical in public health, the food industry and first response. Current processes typically involve laborious cell lysis and genomic DNA extraction to achieve 100–1000 CFU mL⁻¹ levels for detecting gram-positive bacteria. As an alternative to DNA-based methods, cell wall binding domains (CBDs) derived from lysins having a modular structure with an N-terminal catalytic domain and a C-terminal CBD, can be used to detect bacterial pathogens as a result of their exceptionally specific binding to target bacteria with great avidity. We have developed a highly sensitive method for multiplex detection of whole bacterial cells using self-assembled CBD complexes. Self-assembled CBD-SA-reporter complexes were generated using streptavidin (SA), biotin-CBDs, and biotinylated reporters, such as glucose oxidase (GOx) and specific DNA sequences. The simultaneous detection of three test bacteria, Staphylococcus aureus, Bacillus anthracis-Sterne, and Listeria innocua cells in PBS could be accomplished with a 96-well plate-based sandwich method using CBD-SA-GOx complex-coupled spectrophotometric assay to achieve a detection limit of >100 CFU mL⁻¹. To achieve greater detection sensitivity, we used CBD-SA-DNA complexes and qPCR of specific DNA barcodes selectively bound to the surface of target bacterial cells, which resulted in a detection sensitivity as low as 1–10 CFU mL⁻¹ without cross-reactivity. This sensitive multiplex detection of bacterial pathogens using both CBD-SA-GOx and CBD-SA-DNA complexes has the potential to be quickly combined with point-of-care compatible diagnostics for the rapid detection of pathogens in test samples.