Rapid screening of waterborne pathogens using phage-mediated separation coupled with real-time PCR detection

Ziyuan Wang, Danhui Wang, Amanda J. Kinchla, David A. Sela, Sam R. Nugen

Escherichia coli O157:H7 is a ubiquitous pathogen which can be linked to foodborne outbreaks worldwide. In addition to the significant illnesses, hospitalizations, and deaths resulting from the outbreaks, there can be severe economic consequences to farmers, food manufacturers, and municipalities. A rapid detection assay which can validate sanitation and water quality would prove beneficial to these situations. Here, we report a novel bacteriophage-mediated detection of E. coli O157:H7 which utilizes the specific recognition between phages and their host cell as well as the natural lysis component of the infection cycle for DNA release. Carboxylic acid-functionalized magnetic beads were conjugated with bacteriophage and used to separate and concentrate E. coli O157:H7. The effects of bead incubation time, salinity, pH, and temperature on the bio-magnetic separation were investigated and compared to an antibody-based counterpart. The conditions of 0.01 M PBS, pH 7.0, and 20 min of reaction at 37 °C were found to be optimal. The capture efficiency of the coupled assay was approximately 20 % higher than that of antibody-based separation under extreme conditions. The resulting bead-phage-bacteria complexes were quantitatively detected by real-time PCR (qPCR). Our results demonstrated that the use of phage-based magnetic separation coupled with qPCR improved the sensitivity of detection by 2 orders of magnitude compared that without phage-based pre-concentration. Specificity and selectivity of the assay system was evaluated, and no cross-reactivity occurred when Salmonella typhimurium, Staphylococcus aureus, and Pseudomonas aeruginosa were tested. The total assay time was less than 2 h.