Development and Optimization of a Novel Immunomagnetic Separation- Bacteriophage Assay for Detection of Salmonella enterica Serovar Enteritidis in Broth

Abstract

Salmonella is the second-leading cause of food-borne illness in most developed countries, causing diarrhea, cramps, vomiting, and often fever. Many rapid methods are available for detection of Salmonella in foods, but these methods are often insensitive or expensive or require a high degree of technical ability to perform. In this paper we describe development and characterization of a novel assay that utilizes the normal infection cycle of bacteriophage SJ2 for detection of Salmonella enterica serovar Enteritidis in broth. The assay consists of four main stages: (i) capture and concentration of target cells by using immunomagnetic separation (IMS); (ii) infection of the target bacterium with phage; (iii) amplification and recovery of progeny phage; and (iv) assay of progeny phage on the basis of their effect on a healthy population of host cells (signal-amplifying cells). The end point of the assay can be determined by using either fluorescence or optical density measurements. The detection limit of the assay in broth is less than 10 4 CFU/ml, and the assay can be performed in 4 to 5 h. The results of this study demonstrate that the IMS-bacteriophage assay is a rapid, simple, and sensitive technique for detection of Salmonella serovar Enteritidis in broth cultures which can be applied to preenriched food samples.

Maximizing Capture Efficiency and Specificity of Magnetic Separation for Mycobacterium avium subsp. paratuberculosis Cells

“…The combination of phage amplification and MS is not a new concept. Immunomagnetic (IMS)-phage assays for Salmonella enterica serovar Enteritidis and Escherichia coli O157:H7 have been described previously (5,6).…”

Oriented Immobilization of Bacteriophages for Biosensor Applications

“…The interaction between the bacteriophage and the bacterial cell is multivalent, very strong, and specific, making bacteriophages a promising alternative to antibodies for use as the recognition element in a sensor to capture, detect, and identify bacterial species. Phage-based methods for bacterial detection that utilize the following approaches have been developed: (i) monitoring the multiplication of phage particles in the presence of host cells (12,28), (ii) detection of intracellular components released during phagemediated cell lysis (3,15,21), (iii) detection of growth inhibition due to the presence of a specific bacteriophage (23), (iv) use of phages as specific bacterial staining agents (10,14,16), and (v) monitoring the expression of a reporter gene cloned into the phage genome that occurs after bacterial infection (6,15,32). The reported detection techniques based on these approaches were shown to be faster than conventional methods but still did not have sensitivities sufficient to meet the requirements for food and environmental applications.…”

General Interest Scientific and Teechnical Factors Affecting the Setting of Salmonella Criteria for Raw Poultry: A Global Perspective

“…However, PAGE 9 recovering only specific serovars (71,173,186). Due to the poor performance of many selective agars in detecting Salmonella from non-clinical samples, food microbiology laboratories usually use two or more plating media to reduce the likelihood of false negative results (41).…”