Food Protection Trends - July 2008 - (Page 469)

FIGURE 1. The Phast Swab. The bottom of the device contains growth media (Tryptic Soy Broth (TSB) supplemented with 0.2 M glucose) and E. coli O157 specific immunomagnetic (IMS) beads (Invitrogen, Carlsbad, CA). The top contains the reporter bacteriophage and the β-galactosidase substrate. A sampling tool (swab) is also contained in the device. carrying the lacZ gene, bacterial growth medium, E. coli O157:H7 specific immunomagnetic beads and a β-galactosidase substrate (colorimetric or luminescent) within a Snap Valve™ device (Fig. 1). Each component is contained in a separate reservoir and is therefore separated from the other components. Releasing individual components from each reservoir in a stepwise fashion allows for each component to be added to the bottom of the test device in succession, making the entire assay self contained. If a colorimetric substrate is used, the assay can be interpreted visually. Conversely, the use of a luminescent substrate allows for greater sensitivity. In this scenario, the entire test device is placed within a handheld luminometer, which enumerates the photons produced in the luminescent reaction. This novel method has been termed the “Phast Swab” to denote the combination of reporter phage, swabs and speed into an easy-to-use test. In the present study, the Phast Swab was investigated for its ability to detect E. coli O157:H7 on beef samples. MATERIAL AND METHODS Development of the Phast Swab The Phast Swab was developed based on the use of Snap Valve™ devices (Hygiena, Camarillo, CA) (Fig. 2). Briefly, Snap Valve™ devices (Hygiena Corporation, Camarillo, CA) were modified to carry a colorimetric β-galactosidase substrate (chlorophenol red β-D-galactopyranoside (CPRG) Roche, Indianapolis, IN) or a luminescent β-galactosidase substrate (Beta-glo, Promega, Madison, WI) in the cap of the device. The bottom of the device contained growth medium (Tryptic Soy Broth [TSB], Difco, BD, Franklin Lakes, NJ, supplemented with 0.2 M glucose) and E. coli O157specific immunomagnetic separation (IMS) beads (Invitrogen, Carlsbad, CA (Fig. 2A). A sampling device (a swab) was also included. To perform the Phast Swab method, the swab was removed, the surface of the meat was swabbed and the swab was returned to the Snap Valve™ device; an eight-hour enrichment followed. The IMS beads (with any E. coli O157:H7 cells attached) were then concentrated, and the growth medium was removed from the device with a disposable include culture on solid media, immunoassays, and molecular-based (polymerase chain reaction) methods (10). Cultural methods are slow, requiring 24–48 hours for results, while immunoassays and molecular techniques are labor intensive, or time consuming, or they require extensive training of laboratory personnel. In addition, most immunoassays and molecular detection methods cannot distinguish between viable and non-viable cells. Newer detection assays ideally should be designed to detect E. coli O157:H7 on carcasses or beef trim, in the slaughterhouse setting. Reporter bacteriophages (phages) represent a novel alternative for the detection of bacteria within food (5). In this method, DNA carrying a reporter gene is introduced into a target bacterium via a phage. Once the reporter gene has been introduced into the bacterium, it is expressed, thereby allowing bacterial cells to be rapidly identified. Because phages need host cells to replicate, the reporter gene will not be expressed until the phage DNA has been injected into the host. Therefore, expression of the reporter gene is indicative of the presence of the infected organism. A major advantage of using reporter phages to detect bacteria is the ability of the phages to amplify themselves, and in the process, amplify the reporter signal. Also, reporter phages can detect only viable bacteria, which is an advantage over immunological and most genetic detection methods. A vertically integrated assay based on the concept of the reporter phage has been developed that is capable of rapid detection of E. coli O157:H7 (6). The assay combines a sampling device (a swab), an E. coli O157:H7 reporter bacteriophage JULY 2008 | FOOD PROTECTION TRENDS 469

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Food Protection Trends - July 2008 Contents Sustaining Members Lone Star Perspective from Your President Commentary from the Executive Director An Integrated Assay for Rapid Detection of Escherichia coli O157:H7 on Beef Samples Listeria Prevention Practices for Small Cheese Operations New Members News Industry Products Award Winners Committee Meetings Ivan Parkin Lecture John H. Silliker Lecture Preliminary Program Networking Opportunities General Information Registration Form Workshops Exhibitors Special Contributors and Sponsors Coming Events Advertising Index Journal of Food Protection Table of Contents Audiovisual Library Order Form Booklet Order Form Membership Application

Food Protection Trends - July 2008

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