Food Protection Trends - July 2008 - (Page 471)

FIGURE 4. Detection limit of the luminescent Phast Swab assay. An initial inoculum of 101 CFU/100 cm2 gave detectable results. × steps were conducted at 31oC, and following the assay, the Phast Swab was read by use of a SystemSure II handheld luminometer (Hygiena, Camarillo, CA). Evaluation of the Phast Swab E. coli O157:H7 strain 27 (a bovine isolate from our strain collection housed in the Dept. of Animal Sciences at CSU) was used to evaluate the use of the Phast Swab on meat samples (simulated carcass tissue). Stock bacterial cultures were maintained in 30% glycerol and were frozen at -70oC. An overnight culture of E. coli O157:H7 strain 27 was diluted by preparing a series of 10-1 dilutions in lambda buffer until the final dilution was 10 -9. To simulate carcass tissue, ten centimeter by ten centimeter portions of beef (top round, sirloin tip) were inoculated with the serial dilutions of E. coli O157:H7 by pipetting 1 ml of each dilution on an individual 10 × 10 cm (100 cm2) piece of meat and by spreading the dilution over the entire surface of the meat with a glass hockey stick. The concentration of each dilution was determined by plate count. The meat was allowed to dry for one hour, and then the entire surface of each meat sample was swabbed with an individual Phast Swab. The rest of the assay was performed as already described. Two negative controls were included in the assay. The controls consisted of a cell-only control (C1, Fig. 3), which entailed swabbing a meat sample inoculated with E. coli O157:H7 (109 CFU/ml) and completing the assay as already described, except that no reporter phage was added, and a phage-only control (C2, Fig. 3), which entailed swabbing an un-inoculated piece of meat (i.e., meat not previously inoculated with E. coli O157:H7) and completing the assay as already described. Luminescent detection For luminescent detection, the Phast Swab was conducted as described, except that a cell-only control was included for each dilution tested. A phage-only control was also included. Each Phast Swab was read three times, and the readings were subtracted from the mean reading of the cell-only or phage-only control (whichever had the higher RLU reading). Group means for the luminescent readings from each dilution were calculated. For each sample tested, a Student’s t-test was performed, using a Φ value of 0.05. A test result was considered positive if it was 3 standard deviations greater than the corresponding group mean. RESULTS AND DISCUSSION The results of the colorimetric Phast Swab assay are shown in Fig. 3. The visual detection limit was obtained by comparing the test samples to the control samples (C1 and C2). Any sample in which a color change was observed (compared to the controls) was considered a positive test result. With an eight-hour enrichment, the detection limit was an original inoculum of 103 CFU/100 cm2. The results of the colorimetric assay show that the Phast Swab is sensitive, easy to use, and fairly rapid. Further investigation has shown that placing the Phast Swabs in the refrigerator overnight increases the intensity of the color reaction, which helps in situations in which the color reaction is weak. To increase the sensitivity of the assay, we investigated the use of β-galactosidase luminescent substrates. The luminescent substrates display the highest sensitivity and largest dynamic range of any class of β-galactosidase substrate, and these substrates allow the luminescent assay of β-galactosidase to become as sensitive as the assay of bioluminescent luciferase (12). The luminescent assay was based on the use of a coupled luminescent reaction. Coupled assays have been designed using modified forms of luciferin that require the action of a second enzyme to yield luminescence. In this study, a luciferin-galactoside substrate (6-Oβ-galactopyranosylluciferin) (Promega, Madison, WI) produced a detectable signal via a coupled enzyme reaction in which the substrate was first cleaved by β-galactosidase to form luciferin and galactose. The luciferin was then utilized in a firefly luciferase reaction to generate light, which was read by use of a handheld luminometer. When the luminescent substrate was used, the initial 102 CFU/100 cm2 inoculum was easily detectable (T(2) = 297.29, Φ < 0.005), as was the initial 101 CFU/100 cm 2 inoculum (T(2) = 5.586, Φ = 0.031) (Fig. 4). The increased sensitivity of the luminescent substrates indicated that the use of a luminescent substrate should increase the speed of the Phast Swab assay, since a shorter enrichment time would be needed for a positive result. In this study, the use of the luminescent substrate led to detection of E. coli O157:H7 on the beef samples within 10 hours, as opposed to 12 hours with use of the colorimetric substrate, and the limit of detection was 100 fold higher. In previous experiments, the Phast Swab has been tested against other members of the Enterobacteriaceae, including various isolates and strains of non O157:H7 E. coli, Salmonella spp., JULY 2008 | FOOD PROTECTION TRENDS 471

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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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