Phage receptor binding protein-based magnetic enrichment method as an aid for real time PCR detection of foodborne bacteria

Abstract

We present a novel phage receptor binding protein-based magnetic separation and pre-enrichment method as an alternative to the immunomagnetic separation methods by replacing antibodies with bacteriophage receptor binding proteins (RBPs). We couple the proposed RBP-based magnetic separation with real time PCR for rapid, sensitive and specific detection of Campylobacter jejuni cells in artificially contaminated skim milk, milk with 2% fat and chicken broth. Recovery rates, assessed by real time PCR, were greater than 80% for the samples spiked with as low as 100 cfu mL(-1) of C. jejuni cells. The specificity of capture was confirmed using Salmonella Typhimurium as a negative control where no bacteria were captured on the RBP-derivatized magnetic beads. The combination of RBP-based magnetic separation and real time PCR improved PCR sensitivity and allowed the detection of C. jejuni cells in milk and chicken broth samples without a time consuming pre-enrichment step through culturing. The total sample preparation and analysis time in the proposed RBP-based enrichment method coupled with real time PCR was less than 3 h.

Phage‐Based Biosensor and Optimization of Surface Blocking Agents to Detect Salmonella Typhimurium on Romaine Lettuce

“…Many research studies are being conducted to develop a real‐time detection method that can be practical and time‐efficient in the field (Poshtiban et al . ; Seong et al . ; Park et al .…”

Culture‐Independent Rapid Detection Methods for Bacterial Pathogens and Toxins in Food Matrices

“…While many chemical methods and physicochemical methods have been investigated in foodborne separation and concentration (Stevens and Jaykus ), methods based on biological interactions such as immunomagnetic separation (IMS) has become one major approach for separating foodborne bacteria followed by various detection methods (Uyttendaele and others ; Gehring and others ; Ravindranath and others ; Zhao and others ; Yang and others ; Wang and others ). Besides antibody–antigen interaction, ligands such as aptamers (Joshi and others ; Wu and others 2014a), bacteriophage proteins (Poshtiban and others ), and other binding molecules (El‐Boubbou and others ) have been attached to magnetic beads for bacteria or bacterial nucleic acid (Zhang and others ; Li and others 2011b) separation in a similar way as IMS. The magnetic‐based separations have the advantages of specific removal of the target from food particles, inhibitors, and other organisms using rapid and easy manipulation with a common separation time within 1 h (Joshi and others ; Wang and others ; Poshtiban and others ; Cloutier and others ; Wang and others ), good recovery of the target (Uyttendaele and others ; El‐Boubbou and others ; Ravindranath and others ; Yang and others ; Wang and others ), no dilution of the sample or loss of carrier during washing, and possible automation and scale‐up separation (Horák and others ; Lau and others ; Chen and others ).…”

“…Besides antibody–antigen interaction, ligands such as aptamers (Joshi and others ; Wu and others 2014a), bacteriophage proteins (Poshtiban and others ), and other binding molecules (El‐Boubbou and others ) have been attached to magnetic beads for bacteria or bacterial nucleic acid (Zhang and others ; Li and others 2011b) separation in a similar way as IMS. The magnetic‐based separations have the advantages of specific removal of the target from food particles, inhibitors, and other organisms using rapid and easy manipulation with a common separation time within 1 h (Joshi and others ; Wang and others ; Poshtiban and others ; Cloutier and others ; Wang and others ), good recovery of the target (Uyttendaele and others ; El‐Boubbou and others ; Ravindranath and others ; Yang and others ; Wang and others ), no dilution of the sample or loss of carrier during washing, and possible automation and scale‐up separation (Horák and others ; Lau and others ; Chen and others ). The efficiency and selectivity of magnetic separation greatly depends on the ligands, as sometimes it is hard to obtain a ligand with high affinity and specificity to the target.…”

Bacteriophage Based Biosensors: Trends, Outcomes and Challenges

“…The modified seeds were used to extract Campylobacter from milk and chicken samples. In samples infected with 10 2 CFU/mL of Campylobacter cells through RT-PCR, it was a prominent improvement measure of more than 80% in 3 h. To confirm the specific adsorption of phage to Campylobacter, S. typhimurium was used as a negative control [37]. More effort is needed to reach commercial biosensors, given that the initial experimental results indicate that RBPs are capable of being used as diagnostic agents in diagnosing pathogens.…”