ERP Micro December 2019

Test Portion Distribution

Samples were blind-coded and shipped to the collaborating sites at refrigerated temperature (2–8°C). Samples were analyzed by the collaborators on the day of receipt or the day after. Within each shipment, a temperature probe was included. Participants were instructed to return the temperature probe to the coordinating laboratory. In addition, the temperature of one sample (water) was documented upon receipt. Temperature records for each laboratory have been included with the supplementary materials.

Test Portion Analysis

Test portions (25 g) evaluated by the candidate method were enriched with 225 mL of LPT broth, homogenized for 2 min and incubated for 22 h at 37 ± 1°C. Following incubation, a 20 μ L aliquot of each sample was lysed for 5 m at >2000 rpm. Lysed samples were transferred to PCR reaction tubes and assayed on the GENE-UP ® Thermocycler. Regardless of presumptive results, all test portions were confirmed by a direct plating onto Agar Listeria according to Ottavani and Agosti (ALOA). ALOA was incubated at 37 ± 1°C for 24 ± 3 h, and if negative, reincubated for an additional 24 ± 3 h. Isolated colonies from the selective agars were confirmed positive by API Listeria. For test portions evaluated by the reference method, 25 g samples were enriched in HF and incubated for 24 ± 3 h at 30 ± 1°C. The HF was transferred to a secondary enrichment, Fraser Broth (FB), and plated onto Agar Listeria Ottavani and Agosti (ALOA) and PALCAM. FB tubes were incubated at 37 ± 1°C for 48 ± 3 h. ALOA was incubated at 37 ± 1°C for 24 ± 3 h, and if negative, reincubated for an additional 24 ± 3 hours. PALCAM was incubated at 37 ± 1°C for 48 ± 3 h. Isolated colonies from the selective agars were struck to a non-selective agar and confirmed positive using an API Listeria .

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