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

Ji Young Park,Sung-Wook Choi,Tae Jung Park,Hyun-Joo Chang,Min-Cheol Lim,Nari Lee,Gyeongsik Ok,Kisang Park

doi:10.3390/pr8080908

Abstract

In this study, we describe the development of an automated immunomagnetic separation device combined with real-time polymerase chain reaction (PCR) for detecting foodborne bacteria. Immunomagnetic separation (IMS) is a well-known method for the separation and concentration of target bacteria from a large volume of food samples. Magnetic beads functionalized with an antibody provide selectivity for target bacteria such as Escherichia coli O157:H7. Moreover, compared to conventional methods, real-time PCR enables high-sensitivity detection of target bacteria. The method proposed in this study involves three steps: (1) pre-enrichment, (2) automated IMS and concentration of target bacteria, and (3) detection of target bacteria by real-time PCR. Using food samples with a working sample volume as large as 250 mL, the whole process only requires 3 h. As a result, target bacteria in the range of 101–102 colony-forming units per mg or g of sample can be detected in food samples, such as milk, ground beef, and cabbage, by using the proposed approach. We anticipate that the automated IMS system combined with real-time PCR will contribute to the development of a fully automated system for detecting foodborne bacteria and serve as a multi-tester for a variety of bacterial strains in the capacity of a sample-to-answer device in the near future.

Highlights

Escherichia coli O157:H7 is a pathogenic bacterium harmful to human health
We developed an automated immunomagnetic separation (IMS) device for the pretreatment of food samples for selective separation and concentration of target bacteria, and we combined it with real-time polymerase chain reaction (PCR) for quantitative detection of bacteria
After completion of the sample injection, the immunomagnetic beads bound with bacteria were separated automatically and concentrated into a single recovery tube containing 2 mL of PBST (PBS buffer with 0.5% Tween-20)

Summary

Introduction

Escherichia coli O157:H7 is a pathogenic bacterium harmful to human health. The major reservoirs of E. coli O157:H7 include cattle, ground beef, milk, and uncooked vegetables. The magnetism of the magnetic beads can be used to rapidly separate and concentrate bacteria in a large sample volume (250 mL) using an external magnetic field, which can eliminate PCR inhibitors These challenges can be overcome or minimized by the incorporation of IMS during food sample pretreatment [20,21,22,23]. We developed an automated IMS device for the pretreatment of food samples for selective separation and concentration of target bacteria, and we combined it with real-time PCR for quantitative detection of bacteria. The combined system was effective in simplifying the pretreatment steps for the separation and concentration of target bacteria and the elimination of PCR inhibitors, facilitating the detection of low levels of pathogenic bacteria in foods

Materials

Automated IMS Process

Real-Time PCR

Recovery Monitoring and Data Analysis

Establishment of a Standard Curve

Conclusions