Immunosensor-Based Simultaneous Detection of 7 Major STEC Serotypes

Introduction: Shiga Toxin-producing Escherichia coli (STEC), including the most recognized serotype O157,and other major non-O157 STEC serotypes (O26, O45, O103, O111, O121, and O145), commonly known as “Big Six,” have been associated with multiple outbreaks and they are considered as adulterants in certain meat products. Therefore it is highly desirable to develop assays that can simultaneously detect multiple STEC serotypes.

Purpose: The goal of this study was to develop a sensitive and rapid immunosensor-based multiplexing array system to simultaneously detect and identify 7 major STEC serotypes.

Methods:  The mixture of 7 different types of immunosensors, which are pre-encoded magnetic beads functionalized with antibodies to each of 7 STEC serotype, was loaded into a 96-well microplate and used as an array platform. Presence of target STEC was determined and identified by reading fluorescent signals from binding of fluorescently labeled detection antibodies to target STEC cells. The developed array system was tested for its sensitivity and specificity.

Results:  Results from this study showed the developed immunosensor array was an easy-to-operate, high-throughput detection system for multiple STEC serotypes. The array could be completed in less than 5 h. The optimized multiplex array was able to detect STEC as low as 10⁴ CFU/ml without any enrichment and 10-100 CFU/ml with 12 h enrichment. Even though there was cross-reactivity among STECs, each STEC was able to be identified by recognizing unique immunosensor binding pattern.

Significance:  This result indicates the developed immunosensor-based multiplexing array can be reliable method for simultaneous detection of multiple STECs. This array shows great potential to be adapted for automatic testing of food or environmental samples for the presence of STECs.