Detection of Escherichia coli O157:H7 Using Automated Immunomagnetic Separation and Enzyme-Based Colorimetric Assay

Abstract

The food industry requires rapid and simple detection methods for preventing harm from pathogenic bacteria. Until now, various technologies used to detect foodborne bacteria were time-consuming and laborious. Therefore, we have developed an automated immunomagnetic separation combined with a colorimetric assay for the rapid detection of E. coli O157:H7 in food samples. The colorimetric detection method using enzymatic reaction is fascinating because of its simplicity and rapidity and does not need sophisticated devices. Moreover, the proposed procedures for the detection of bacteria in food take less than 3 h including pre-enrichment, separation and detection steps. First, target-specific immunomagnetic beads were introduced to contaminated milk in a pre-enrichment step. Second, the pre-enriched sample solution containing target bacteria bound on immunomagnetic beads was injected into an automated pretreatment system. Subsequently, the immunomagnetic beads along with target bacteria were separated and concentrated into a recovery tube. Finally, released β-galactosidase from E. coli O157:H7 after lysis was reacted with chlorophenol red β-galactopyranoside (CPRG) used as a substrate and the colorimetric change of CPRG was determined by absorbance measuring or the naked eye. By the proposed approach in this study, we could detect 3 × 102 CFU/mL of E. coli O157:H7 from a milk sample within 3 h.

Detection of E. coli O157:H7 in Food Using Automated Immunomagnetic Separation Combined with Real-Time PCR

“…In order to confirm the efficacy of the IMS system compared with the real-time PCR analysis, 1/10 of the recovered solution containing the target bacteria was subjected to cell counting. The performance and efficiency of the automated IMS device developed in our study have been confirmed by previous studies [26,27]. It has been proposed in previous literature that the efficiency should be between 90% and 110% in order for it to be considered suitable for quantitative detection [28].…”

“…The principle of the automated IMS operation and the performance of the fabricated automated IMS device have been described in our previous studies [26,27]. In brief, the automated IMS device used consisted of a peristaltic-pump-based fluidics control component and an external magnetic field-based bacterial recovery component.…”

“…These results were obtained by real-time PCR under the same operating conditions used for the recovered sample. Thus, it was possible to quantify the cells in the recovered sample and to theoretically evaluate the initial concentration of E. coli O157:H7 present in the foods [27].…”

Amperometric Biosensors as an Analytical Tool in Food, Dairy and Fermentation Industries

“…Available online at www.globalresearchonline.net hence detection of E. coli. The biosensor was tested in dilute milk samples and results were correlated with traditional methods like PBS buffer 86 and Plate count method 87 and the output was in deviation in the range of 10%. Experiments have been conducted and reported showing that the biosensor has high specificity and selectivity towards the target pathogen even in mixed culture.…”

Immunomagnetic separation: An effective pretreatment technology for isolation and enrichment in food microorganisms detection

“…In recent years, a growing number of studies have focused on enhancing the detection sensitivity and reducing the testing time from days to hours. The representative methods include immunology‐based methods (enzyme linked immunosorbent assay, ELISA; immunochromatographic assay, ICA), nucleic acid‐based methods (polymerase chain reaction, PCR; loop‐mediated isothermal amplification, LAMP), signal‐based methods (such as fluorochrome and quantum dots [QDs]), and so on (Bi et al., 2020; Li et al., 2017; Li et al., 2019; Park, Park, Ok, Chang, & Lim, 2020; Wei et al., 2019; Xue, Zheng, Zhang, Jin, & Lin, 2018). However, food samples are usually complex and heterogeneous systems.…”