General detection of Shiga toxin 2 and subtyping of Shiga toxin 1 and 2 in Escherichia coli using qPCR

Abstract Shiga toxin-producing E. coli (STEC) is a gastrointestinal pathogen and has been recognized as one of the serious problems in public health. Shiga toxin genes ( stx ) can be grouped into different types according to their differences in sequence and biological activities. The two main groups of stx are stx 1 and stx 2 with each group containing several subtypes. It is essential to develop rapid stx 1 / stx 2 subtyping assays and accurate stx 2 general detection assays to provide a quicker turn-around time to predict disease outcome and also to provide data for surveillance purposes. The stx 2 general detection qPCR assay developed in this study showed 100% sensitivity and 100% specificity with no cross reactivity with stx 2 negative STEC and non-STEC isolates. This stx 2 general detection assay was able to detect all seven different stx 2 subtypes at low level of detection and with good PCR efficiency. In addition, stx 1 / stx 2 subtyping qPCR assays were succesfully developed to detect all stx 1 subtypes and stx 2 subtypes with the exception of one stx 2b subtype carried by one strain. The qPCR stx 1 / stx 2 subtyping assays showed 100% specificity with no cross reactivity on subtypes not targeted by each assay. The rapidity with faster turn-around time along with high throughput volume of the stx 1 / stx 2 subtyping and stx 2 general detection qPCR assays will have great value as tools for STEC associated risk assessment, outbreak monitoring, epidemiology studies, and clinical management.