Committee on microbiology and extraneous materials

Journal of AOAC International, Jan-Feb, 2008 by Wayne A. Ziemer, Melissa C. Newman, Todd Marrow, James R. Agin, Michael H. Brodsky, James E. Brown, Joseph L. Ferreira, Yvonne M. Hale, Walter E. Hill, Charles E. Pixley, Rice Daniel, Roxanne Shively, Kevin J. Vought, Robert A. LaBudde, Robert W. Phillips, Paul Wehling

For ruggedness testing, the following method parameters were investigated: (1) primary enrichment temperature (40, 42, and 44[degrees]C); (2) the use of autoclaved primary enrichment media base and boiled secondary media; (3) sample volume (0.5, 1.0, 1.1, and 1.25 mL); (4) operating temperature of the test strip assay (4, 15, room temperature, and 37[degrees]C); and, (S) variations in time of reading the test strip (immediately, 5, 10, and 20 min). Results demonstrated that method performance was not affected by variation in the parameters with the exception of the operating temperature of the test strip. Test strip results were equivalent at temperatures from 15-37[degrees]C. For test strips that were run at 4[degrees]C, the liquid sample moved more slowly through the test strip matrix resulting in the development of test signals that were lighter in intensity than test strips that were run at temperatures [greater than or equal to]-15[degrees]C. However, at 4[degrees]C, the ultimate test result was not affected.

(3) VIDAS[R] Listeria species Xpress (LSX) with Ottaviani Agosti Agar, Matrix Extension: The VIDAS Listeria species Xpress (LSX; bioMerieux, Inc., 595 Anglum Rd, Hazelwood, MO 63042-2320) test is an enzyme-linked fluorescent immunoassay (ELFA) designed for use with the automated VIDAS or mini-VIDAS instruments for the specific detection of Listeria species. Meat and dairy products require 2-step 30 h enrichments in proprietary LX broth. Environmental swabs and sponges require 1-step 24 h enrichments in LX broth. The Solid Phase Receptacle (SPR) serves as the solid phase as well as the pipetting device for the assay. The SPR is coated with anti-Listeria antibodies. Reagents for the assay are ready-to-use and predispensed in the sealed reagent strips. The instrument performs all of the assay steps automatically. The user places the sample into the reagent strip. Then the sample is cycled in and out of the SPR for a specific length of time. Listeria antigens present in the sample will bind to the anti-Listeria monoclonal antibodies, which are coated on the interior of the SPR. Unbound sample components are washed away. Alkaline phosphatase-labeled antibodies are cycled in and out of the SPR and will bind to any Listeria antigens captured on the SPR wall. Further wash steps remove unbound conjugate. During the final detection step, the substrate (4-methyl-umbelliferyl phosphate) is cycled in and out of the SPR. The bound enzyme conjugate catalyzes the hydrolysis of this substrate into a fluorescent product (4-methyl-umbelliferone), the fluorescence of which is measured at 450 nm. When the assay is completed, the results are analyzed automatically by the instrument and a test value is generated for each sample. This value is then compared to an internal reference (threshold) and each result is interpreted (positive, negative).

For the inclusivity study, all 50 Listeria strains gave positive results. For the exclusivity study, all 70 non-Listeria species gave negative results.