Detection of shiga toxins by lateral flow assay

Abstract

Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA) for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD) for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing.

Figure 1

Detection of shiga toxins from STEC serotypes by lateral flow assay (LFA). Supernatant from Stx1 and seven variant Stx2-producing STEC cultures were diluted 1:5 in PBS and evaluated using the Stx LFA. −/−, a non-Stx-producing E. coli serotype; LB, Luria broth; T, test line; C, control line.

Figure 2

Dose-dependent detection of purified Stx2a by LFA. Purified Stx2a was serially diluted in PBS from 500 to 0.1 ng/mL and assessed by LFA (top panel); Density measurements were obtained at the test line from three independent experiments and the data plotted as the mean (±SEM) at the test line for each Stx2a dilution (bottom panel). Regression analysis revealed a linear relationship between the density of the test line and the concentration of Stx2a between 20 and 2.5 ng/mL (R² = 0.99). T, test line; C, control line.

Figure 3

Detection of Stx2a from spiked milk matrices by LFA. Samples of nonfat, 1% and 2% milk were spiked with purified Stx2a (50–0.1 ng/mL) and centrifuged to remove lipids. The defatted samples were then diluted 10-fold in PBS and evaluated for Stx2a detection by LFA. T, test line; C, control line; +, positive test result; −, negative test result.

Figure 4

Detection of Stx2a from spiked lettuce samples by LFA. Lettuce samples were chopped, suspended in PBS and spiked with purified Stx2a (100–1 ng/mL). Samples were then mixed, centrifuged and the resulting supernatants evaluated for Stx2a detection using the LFA. T, test line; C, control line; +, positive test result; −, negative test result.

Figure 5

Detection of Stx2a from spiked ground beef by LFA. Ground beef was suspended in PBS and spiked with purified Stx2a (100 to 1 ng/mL). The samples were mixed, centrifuged to reduce fat content and the resulting supernatant evaluated for Stx2a detection by LFA (top panel). Density measurements were obtained at the test line from three independent experiments and data plotted as the mean (±SEM) at the test line for each Stx2a dilution (bottom panel). The dotted line represents the average density at the test line for beef without an Stx2a spike plus three standard deviations. T, test line; C, control line.