Phage-Displayed Peptide-Q Dot Nanocrystal Combo for High-Sensitivity Bacterial Detection in Plasma.

Despite improved phlebotomy practices, refrigeration of red cells, freezing of plasma and improved materials for transfusion product collection and storage, bacterial contamination of transfusion products is still a longstanding problem. Current bacterial detection tests relevant to transfusion medicine, especially for stored platelets have limitations with regard to time, specificity and sensitivity. There is a need for new and improved cost-effective high-affinity detection probes to fill this gap. In this study, using plasma spiked with Bacillus cereus 4342 and B. anthracis-Sterne as an experimental system, we identified peptides from a bacteriophage-displayed random peptide library that selectively bind and detect the Bacillus strains. By labeling the peptides with Q dot-liquid nanocrystals, the detection sensitivity of the peptides was further enhanced.

A commercially available bacteriophage-displayed random peptide library was screened using B. cereus 4342 as bait, using appropriate controls under stringent conditions. The screening and subsequent sequencing of the phage DNA identified two phages each containing a coding sequence for 12-amino acid peptide that are selectively capable of binding to the Bacillus. Based on the nucleic acid sequence, the two synthetic peptides with biotin tag were prepared for detection assays and to enhance the detection sensitivity further, the peptides were labeled with streptavidin-conjugated fluorescent quantum-dots (Q dots). Fluorescence was measured either by a plate reader or using a fluorescence microscope.

The two synthetic peptides selectively bound to the Bacillus strains in both dot blot and ELISA assays. The membrane-based dot blot assay demonstrated an assay sensitivity of 10³ colony forming units/ml (CFU/ml), whereas ELISA demonstrated a sensitivity of 10² CFU/ml detection limit. Fluorometry analysis of spiked plasma samples revealed that the two peptides were able to bind to B. cereus 4342 and B. anthracis Sterne and detect these bacteria in plasma at 10² CFU/ml concentrations. The peptide-Qdot combo even detected a single bacterium in fluorescence microscopy.

Overall, the results reported here validate the usefulness of affinity-selected recombinant filamentous phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity detection probes for bacteria in various platforms and settings relevant to the blood safety and transfusion medicine.

The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

No relevant conflicts of interest to declare.