Escherichia coli
O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for
E. coli
O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for
E. coli
O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of
E. coli
O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing
E. coli
. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy.
Efficient phage synthesis yields O157 antigen-specific phages with the detection tag at optimal sites, enabling specific detection of diverse
E. coli
O157 clinical isolates and suggesting broad applications in bacterial detection and phage therapy.