Same-day detection of Escherichia coli O157:H7 from spinach by using electrochemiluminescent and cytometric bead array biosensors

Abstract

Contamination of fresh produce with Escherichia coli O157:H7 and other pathogens commonly causes food-borne illness and disease outbreaks. Thus, screening for pathogens is warranted, but improved testing procedures are needed to allow reproducible same-day detection of low initial contamination levels on perishable foods, and methods for detecting numerous pathogens in a single test are desired. Experimental procedures were developed to enable rapid screening of spinach for E. coli O157:H7 by using multiplex-capable immunological assays that are analyzed using biosensors. Detection was achieved using an automated electrochemiluminescent (ECL) assay system and a fluorescence-based cytometric bead array. Using the ECL system, less than 0.1 CFU of E. coli O157:H7 per gram of spinach was detected after 5 h of enrichment, corresponding to 6.5 h of total assay time. Using the cytometric bead array, less than 0.1 CFU/g was detected after 7 h of enrichment, with a total time to detection of less than 10 h. These results illustrate that both biosensor assays are useful for rapid detection of E. coli O157:H7 on produce in time frames that are comparable to or better than those of other testing formats. Both methods may be useful for multiplexed pathogen detection in the food industry and other testing situations.

FIG. 1.

E. coli O157:H7 detection in buffer and spinach rinse by using the automated ECL assay. E. coli O157:H7 was spiked into either PBS buffer or spinach rinse at increasing concentrations. (A) Raw ECL signals. n is 9 for PBS and 6 for spinach rinse samples. Error bars represent standard deviations but are indistinguishable from the symbols due to their small size. (B) Signal-to-noise (S/N) ratios were calculated by dividing the average signal for each concentration across all experiments by the average signal for the unspiked sample blank across all experiments. Encircled points indicate the lowest spike concentration exhibiting positive detection.

FIG. 2.

E. coli O157:H7 detection in buffer by using the IMS-CBA assay. Heat-killed E. coli O157:H7 was diluted in PBST at increasing concentrations. (A) Median fluorescence intensities. (B) Signal-to-noise (S/N) ratios were calculated versus the unspiked sample. The encircled point indicates the lowest spiked concentration resulting in positive detection. n is 13 for blanks and 9 for all other samples. Error bars represent standard deviations between experiments.

FIG. 3.

E. coli O157:H7 detection in enriched spinach rinse by using the automated ECL assay. E. coli O157:H7 was spiked into spinach rinse and enriched in mBPWp. Signal-to-noise (S/N) ratios were calculated versus the filtered control. Samples were analyzed in triplicate in three independent experiments. Error bars represent standard deviations between experiments.

FIG. 4.

E. coli O157:H7 detection in enriched spinach rinse by using the IMS-CBA assay. E. coli O157:H7 was spiked into spinach rinse and enriched in mBPWp. Signal-to-noise (S/N) ratios were calculated versus the filtered control. Samples were analyzed in triplicate in three independent experiments. Error bars represent standard deviations between experiments.

FIG. 5.

Correlation of ECL signal with E. coli O157:H7 target cell concentration. ECL signals generated from both spiked spinach wash and spiked spinach leaves were compared against concentrations of E. coli O157:H7 in enrichment samples. (A) E. coli O157:H7 concentration versus ECL signal for spiked spinach rinse. (B) E. coli O157:H7 concentration versus ECL signal for spiked spinach leaves. Encircled clusters indicate correlated groupings. Data points represent the replicate average for each concentration within each experiment (n = 3).