Purpose: The purpose of this study was to evaluate droplet digital PCR as a method to unambiguously identify STEC by detecting and co-localizing genomic markers.
Methods: A culture of E. coli characterized by the presence of the two genomic markers, O26 and eae, was enumerated on TCS plates and partitioned into nanoliter volume droplets. Droplets were submitted to various treatments including heating, enzymatic treatment, and exposure to antibacterial peptides, intended to lyse encapsulated bacteria. Duplex PCR amplification of both O26 and eae markers was performed and results were analyzed according to a linkage detection method based on the observation that presence of co-linear markers will increase the number of double-positive droplets relative to the number expected due to chance. A control experiment was run with a mixture of strains bearing one marker.
Results: Bacteria enumeration obtained by PCR positive droplet counting yielded a result matching the initial plate enumeration. Moreover, while no linkage was observed between genes from independent strains, mathematical analysis indicated a significant linkage for double positive bacteria. Optimization of the in-droplet lysis allowed to recover >90% of linkage, thereby demonstrating that ddPCR was able to adequately detect bacteria displaying co-linear makers.
Significance: These results suggest that ddPCR may be a straightforward method to detect in a single PCR run the presence of bacteria such as pathogenic STEC characterized by the concomitant presence of several markers.