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

摘要

Shiga toxin-producing E. coil (STEC) is a gastrointestinal pathogen and has been recognized as one of the serious problems in public health. Shiga toxin genes (six) 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 sbci/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.