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

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.

Bacteriophage receptor binding protein based assays for the simultaneous detection of Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of foodborne gastroenteritis which is occasionally followed by a debilitating neuropathy known as Guillain-Barré syndrome. Rapid and specific detection of these pathogens is very important for effective control and quick treatment of infection. Most of the diagnostics available for these organisms are time consuming and require technical expertise with expensive instruments and reagents to perform. Bacteriophages bind to their host specifically through their receptor binding proteins (RBPs), which can be exploited for pathogen detection. We recently sequenced the genome of C. jejuni phage NCTC12673 and identified its putative host receptor binding protein, Gp047. In the current study, we localized the receptor binding domain to the C-terminal quarter of Gp047. CC-Gp047 could be produced recombinantly and was capable of agglutinating both C. jejuni and C. coli cells unlike the host range of the parent phage which is limited to a subset of C. jejuni isolates. The agglutination procedure could be performed within minutes on a glass slide at room temperature and was not hindered by the presence of buffers or nutrient media. This agglutination assay showed 100% specificity and the sensitivity was 95% for C. jejuni (n = 40) and 90% for C. coli (n = 19). CC-Gp047 was also expressed as a fusion with enhanced green fluorescent protein (EGFP). Chimeric EGFP_CC-Gp047 was able to specifically label C. jejuni and C. coli cells in mixed cultures allowing for the detection of these pathogens by fluorescent microscopy. This study describes a simple and rapid method for the detection of C. jejuni and C. coli using engineered phage RBPs and offers a promising new diagnostics platform for healthcare and surveillance laboratories.

Recent advances in bacteriophage based biosensors for food-borne pathogen detection.

Foodborne diseases are a major health concern that can have severe impact on society and can add tremendous financial burden to our health care systems. Rapid early detection of food contamination is therefore relevant for the containment of food-borne pathogens. Conventional pathogen detection methods, such as microbiological and biochemical identification are time-consuming and laborious, while immunological or nucleic acid-based techniques require extensive sample preparation and are not amenable to miniaturization for on-site detection. Biosensors have shown tremendous promise to overcome these limitations and are being aggressively studied to provide rapid, reliable and sensitive detection platforms for such applications. Novel biological recognition elements are studied to improve the selectivity and facilitate integration on the transduction platform for sensitive detection. Bacteriophages are one such unique biological entity that show excellent host selectivity and have been actively used as recognition probes for pathogen detection. This review summarizes the extensive literature search on the application of bacteriophages (and recently their receptor binding proteins) as probes for sensitive and selective detection of foodborne pathogens, and critically outlines their advantages and disadvantages over other recognition elements.