A Four-Plex Real-Time PCR Assay, Based onrfbE,stx1,stx2, andeaeGenes, for the Detection and Quantification of Shiga Toxin–ProducingEscherichia coliO157 in Cattle Feces

Abstract

Several real-time polymerase chain reaction (PCR) assays have been developed to detect and quantify Shiga toxin-producing Escherichia coli (STEC) O157:H7, but none have targeted the O-antigen specific gene (rfbEO157) in combination with the three major virulence genes, stx1, stx2, and eae. Our objectives were to develop and validate a four-plex, quantitative PCR (mqPCR) assay targeting rfbE(O157), stx1, stx2, and eae for the detection and quantification of STEC O157 in cattle feces, and compare the applicability of the assay to detect STEC O157 to a culture method and conventional PCR (cPCR) targeting the same four genes. Specificity of the mqPCR assay to differentially detect the four genes was confirmed with strains of O157 and non-O157 STEC with different profiles of target genes. In cattle feces spiked with pure cultures, detection limits were 2.8×10(4) and 2.8×10(0) colony-forming units/g before and after enrichment, respectively. Detection of STEC O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. The mqPCR detected 48.9% (136/278) of samples as positive for E. coli O157. Of the 100 samples that were randomly picked from 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar's chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect STEC O157 in cattle feces. However, the use of real-time PCR as a screening method to identify positive samples and then subjecting only positive samples to a culture method may underestimate the presence of STEC O157 in fecal samples.

Occurrence and quantification of Shiga toxin-producing Escherichia coli from food matrices

“…Development of quantification methods for bacterial cells in food matrices aids in the screening of food samples for the presence of potential pathogens and aids in detecting the level of contamination. The accurate quantification of stx 1 gene carrying STEC can be accomplished by real-time quantitative qPCR method [ 7 , 11 ]. The study aims to detect STEC and to develop a qPCR assay to quantify the bacterial DNA present in different food matrices.…”

Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces

“…A culture-independent method, such as quantitative polymerase chain reaction (qPCR), is often applied to quantify STEC in feces [ 11 ]. However, this method requires a DNA extraction and the limit of quantification is higher (10 3 –10 4 CFU/g) compared to the culture-dependent techniques [ 6 , 9 , 12 , 13 , 14 ]. Furthermore, this approach is based on relative quantification and totally dependent on the accuracy of the standard curve construction [ 15 ].…”

A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses

“…cons through DNA electrophoresis (Cao et al, 2005;Giammarioli et al, 2008;Huang et al, 2009;Lee et al, 2007;Li et al, 2007;Liu et al, 2013). These procedures involve extra efforts for post-PCR processing, and the sensitivity is generally one log lower than realtime PCR (Jacob et al, 2012;Noll et al, 2015). Multiplex real-time PCR or multiplex real-time RT-PCR has been increasingly used for high throughput testing with faster turnaround.…”