Validation study to demonstrate the equivalence of a minor modification of Assurance® Enzyme Immunoassay Method, official method 996.14, for detection of Listeria in foods and environmental surfaces to the reference culture methods

Journal of AOAC International,  Jan-Feb, 2008  by Philip T. Feldsine,  David E. Kerr,  Andrew H. Lienau

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The Assurance[R] Enzyme Immunoassay (EIA) for the Detection of Listeria in Foods and Environmental Surfaces, AOAC Official Method 996.14, has been modified to combine the separate antibody and conjugate addition steps into one. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture methods. Two food matrixes and one environmental surface were analyzed. In total, there were valid results from 145 samples and controls. Results showed that the Assurance EIA for Listeria spp. is equivalent to the reference culture methods for the detection of Listeria.

A methods comparison study was conducted to demonstrate the equivalence of a minor modification to l iAOAC Official Method 996.14, Assurance Enzyme Immunoassay (EIA) Method for the Detection of Listeria in Foods and Environmental Surfaces, to the reference culture methods. The method uses highly specific antibodies directed against antigens produced by Listeria. Assurance EIA was adopted First Action in 1996. Method applicability was extended to include environmental surfaces in 2001.

The existing EIA method incorporates 3 steps in the assay protocol--an antibody addition step, a conjugate addition step, and a substrate addition step. A minor modification has been developed that combines the antibody and conjugate addition steps into one. The modified method is more streamlined, resulting in time and labor savings desired by laboratories today. This minor modification was compared to the reference culture methods.

Experimental

Design

A methods comparison study was conducted to evaluate the modified EIA method to the reference culture methods for selected foods and environmental surfaces. Specifically, the foods and surface to be analyzed were raw shrimp, ice cream, and stainless steel. For stainless steel, the reference method used for comparison was the U.S. Department of Agriculture (USDA) culture method (1). For raw shrimp and ice cream, the reference method used for comparison was the U.S. Food and Drug Administration [FDA; Bacteriological Analytical Manual (BAM)] culture method (2). The Listeria species used to inoculate food types/surface are indicated in Table 996.14.

The foods and surface were inoculated with Listeria to achieve 2 levels, a low level where fractional recovery was anticipated and a high level where predominantly positive results were expected. For each food/surface, 20 replicate test portions representing the low and 20 replicate test portions representing the high contamination levels were analyzed by the EIA method and the reference culture methods. Five uninoculated test portions were also analyzed by each method.

Preparation of Inocula and Materials

Foods were initially screened for natural contamination by Listeria. Because no natural contamination was found, the foods were artificially contaminated with the target organism.

Strains used as sources of inocula were grown in brain heart infusion (BHI) broth for 18-24 h at 35-37[degrees]C. Foods received inocula of stationary phase cells estimated to achieve 2 seed levels: 1-5 colony-forming units [CFU; most probable number (MPN)]/25 g (low) and 10-50 MPN/25 g (high), when measured by MPN at the time of sample analysis. Fractional response rate was expected from at least one of the 2 levels. Raw shrimp and ice cream were stored frozen at -20[degrees]C for at least 2 weeks prior to analysis. MPN procedures were conducted on the day of initiation of analyses and were used to estimate the number of target organism/g for each food. MPNs were determined by using 3 replicates of 100, 10, 1, and 0.1 g samples and evaluated using the BAM or USDA specified procedures. MPN was calculated using the dilution tables available in the BAM.

For environmental samples, the strain was grown in BHI broth for 18-24 h at 35-37[degrees]C. The initial culture was diluted into 10% nonfat dry milk (w/v; NFDM) in Butterfield's phosphate buffered diluent (BPBD) to a volume that allowed for even distribution of inoculum over the entire test surface area, but without producing excessive accumulation of liquid that might dry unevenly. The surface type received an inocula of cells sufficient to provide fractional recovery. The surface was allowed to dry for 16-48 h at room temperature (20-25[degrees]C). The surface was visually dry at the time of test portion collection.

Test Portion Analysis and Confirmation

Each EIA method test portion was tested by the EIA method according to the manufacturer's package insert. All test portions, for both the EIA method and reference methods, were confirmed according to the appropriate culture method. Using the selective agar plates, isolates were biochemically identified using MicroID strips (Remel, Lenexa, KS).

Data Analysis

Because the test method and the culture method use a different primary enrichment, statistical analyses for method comparison of all foods were performed following the Mantel-Haenszel technique for unmatched samples (3). For all comparisons, a Chi square value >3.84 is indicative of a significant difference at the 95% probability level. All performance statistics were performed on a per-level and per-food basis.