A qPCR assay to detect and quantify Shiga toxin-producing E. coli (STEC) in cattle and on farms: a potential predictive tool for STEC culture-positive farms

Abstract

Shiga toxin-producing E. coli (STEC), of various serogroups harboring the intimin gene, form a serious threat to human health. They are asymptomatically carried by cattle. In this study, a quantitative real-time PCR (qPCR) method was developed as a molecular method to detect and quantify Shiga toxin genes stx1 and stx2 and the intimin gene eae. Subsequently, 59 fecal samples from six farms were tested using qPCR and a culture method as a reference. Three farms had contaminated animals as demonstrated by the culture method. Culture-positive farms showed moderate significantly higher stx prevalences than culture-negative farms (p = 0.05). This is the first study which showed preliminary results that qPCR can predict STEC farm contamination, with a specificity of 77% and a sensitivity of 83%, as compared with the culture method. Furthermore, the presence or quantity of stx genes in feces was not correlated to the isolation of STEC from the individual animal. Quantitative data thus did not add value to the results. Finally, the detection of both stx and eae genes within the same fecal sample or farm using qPCR was not correlated with the isolation of an eae-harboring STEC strain from the respective sample or farm using the culture method.

Figure 1

Quantification of stx1, stx2 and eae genes by qPCR in cattle fecal samples artificially inoculated with STEC cells. Artificial inoculation was performed using various dilutions of strain MB3936 in 16 contamination levels (a) and of strain MB4378 in five contamination levels (b). Primer-probe set a was used to quantify stx2 gene of strain MB3936 and primer-probe set b to quantify stx2e gene of strain MB4378.

Figure 2

Quantification of stx1, stx2 and eae genes by qPCR in individual cattle fecal samples (n = 59). (a) Samples originated from three culture-positive farms (A, B, C) and (b) three culture-negative farms (D, E, F). * Cattle fecal samples that were found to be culture-positive. Primer-probe sets a and b were used to quantify all possible stx2 variants.