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

The method was modified by changing the enrichment for environmental surfaces from a 2-step to a single-step procedure. The current version specifies a 2-stage enrichment procedure, first in half-Fraser broth and then in buffered Listeria enrichment broth, with a total incubation time of 42 to 48 h. The new single-step enrichment procedure, for environmental samples, utilizes Listeria enrichment single step (LESS) broth and has an enrichment period of as little as 25 h.

For the inclusivity study, all 52 Listeria strains gave positive results. Exclusivity testing was performed in the original PTM study (960701).

Results from the internal laboratory methods comparison study showed that the Reveal method was more productive (P < 0.05) than the reference USDA culture procedure (3) for 2 surfaces (stainless steel and plastic), and statistically comparable to the reference method for the other 3 surfaces (cast iron, ceramic tile, and sealed concrete). Overall, sensitivity of the Reveal method at 24 h compared to plating from LESS broth to Oxford agar was 87%, while compared to the USDA culture procedure the Reveal method detected 74% more positive samples. An independent laboratory trial with ceramic tile inoculated with L. monocytogenes confirmed the effectiveness of the Reveal method at the 24 h time point. Of a total of 40 inoculated surface samples at 2 levels, the Reveal method produced 34 positives (vs 38 by plating from LESS broth to Oxford agar for a sensitivity of 89%). This compared favorably to the 28 positives detected by the USDA reference culture procedure. There were no false-positive results with the Reveal test in either the internal or independent laboratory trials, therefore the test specificity was 100%. The new, abbreviated enrichment procedure for environmental samples allows results to be obtained in as little as 25 h from sample collection. The enrichment procedure for food samples remains as previously described.

Ruggedness testing was not performed for this study.

(5) DuPont Qualicon BAX[R] System PCR Assay for Screening Listeria genus: Q7 BAX[R] Instrument Modification: The BAX[R] System (Qualicon, Inc., ESL Bldg 400, Rt 141 and Henry Clay, Wilmington, DE 19880) uses PCR to amplify a specific fragment of bacterial DNA, which is stable and unaffected by growth environment. The fragment is a genetic sequence that is unique to the genus Listeria, thus providing a highly reliable indicator that the organism is present. The BAX system measures the denaturation temperature and analyzes the magnitude of the fluorescent signal change to determine a positive or negative result. The system combines primers, polymerase, and nucleotides needed for PCR into a single tablet. The specificity of a PCR assay is determined by the DNA sequences of the primers used.

Most chemical and operational parameters of the new instrument/assay kit combination are the same with the new instrument as with the current instrument. All currently available assays will be compatible with both instruments. All kit reagents are identical to those used in the kits previously AOAC validated. All components of the BAX tablets including PCR primers, Taq, nucleotides and bulking agents are the same. Kits are thus fully compatible using both the current BAX and Q7 instruments. The only operational difference between the 2 instruments is in the way that the signal is recorded. Emission of bound SYBR Green is detected using a photomultiplier tube in the current BAX platform. In the Q7 instrument, a charged coupled device camera is used to detect the emitted light.