AOAC-RI ERP Micro June 2016

OMAMAN-29 D/ PTM Validation Report 111501 OMA ERP - June 2016 ERP Use Only

Results

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All 55 Listeria strains were detected by the 3M MDA 2 - Listeria method. L. grayi recovery was enhanced when a food sample (Ultra-high-temperature [UHT] milk as described in ISO 16140 [9]) was added to the medium in the standard 1:10 dilution scheme. None of the 30 non- Listeria

strains were detected. See Tables 2 and 3 for study details and results.

Matrix Study

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The method comparison study consisted of evaluating a total of 30 un-paired sample replicates for 10 matrixes, along with naturally contaminated raw chicken (leg pieces), tested at the independent laboratory, evaluating 20 sample replicates using two separate lots, see Table 4a. The raw chicken (leg pieces and fillets) analyzed at the internal lab was inoculated according to AOAC guidelines which are described below, see Table 4b. Within each sample set, there were 5 uninoculated samples (0 CFU/test portion), 20 low level inoculated samples (0.2-2 CFU/test portion), and 5 high level inoculated samples (2-5 CFU/test portion), except for the naturally contaminated raw chicken (leg pieces). The inoculum was prepared by transferring a single Listeria colony from Trypticase Soy Agar with 5% Sheep Blood (SBA) into BHI broth and incubating the culture at 35 ± 2 o C for 24 ± 2 hours. Tables 4a and b presents the sample

preparation guidelines for the matrix.

All matrixes were screened for the presence of the target organism following the appropriate reference method. Additionally, an aerobic plate count (APC) was conducted following the FDA/BAM Chapter 3 reference [10] method to determine the level of background flora in each

test matrix prior to inoculation.

Prior to inoculation of beef hot dogs, deli turkey, and queso fresco the broth culture inoculum was heat stressed for 10 ± 1 minute at 50 ± 1°C in a water bath. The degree of injury of the culture was estimated by plating an aliquot of diluted culture onto Modified Oxford Agar (MOX) and Tryptic Soy Agar (TSA). The agars were incubated at 35 ± 1°C for 24 ± 2 hours and the

100 ) AOAC Research Institute Expert Review Panel U e Only 1( x n n nonselect select − = number of colonies on selective agar and n nonselect = number of colonies on non-

colonies were counted. The degree of injury was estimated as:

Where n select

selective agar. Using BHI broth as the diluent, the culture was diluted to a low level expected to yield fractional positive results (5-15 positive results) and a high level expected to yield all positive results. Following inoculation, a bulk lot of the matrix was homogenized by hand and held for 48-72 hours at refrigerated temperature (2-8 °C) prior to analysis to allow time for the

organism to equilibrate within the sample.

For the inoculation of the whole melons, a single whole melon was placed into a large sterile bag and the blossom end of the melon was inoculated with 100 µL of the diluted Listeria monocytogenes culture. The liquid culture was then allowed to soak into the melon. The melon