ERP Micro December 2019

GENE-UP ® L. monocytogenes 2 (LMO 2)

050568 - 02 - 2018-12 - en

2. Follow the same procedure in the FINAL SETUP FOR PCR section of this document, using 10 μL of this dilution of lysate.

Note: It is recommended to retest in parallel the lysate without dilution. Note: In case of inhibited results at 1:3, you can dilute the lysate to 1:10.

Note: Some matrices may contain inhibitory molecules. For such difficult matrices, 1 mL of enriched sample could be diluted 1:10 in Tryptone Salt or Normal Saline prior to lysis step. If inhibition persists, proceed to an additional 1:3 dilution as described above. CONFIRMATION OF POSITIVE RESULTS Confirmation of Positive Results Obtained Using the Method Certified NF VALIDATION or the Protocol for Feed Samples In the context of NF VALIDATION mark, all positive results obtained must be confirmed. Confirmation must be performed using the enrichment broth stored at +2°C/+8°C and mixed thouroughly by hand, and must be initiated within 72 hours following the end of the incubation period. Follow one of the validated procedures below: 1. Isolate the enriched sample, and streak 10 μL on chromogenic agar according to the definition of EN ISO 11290 (Ottaviani Agosti formulation type) or forming part of an ISO 16140 certified method. The presence of characteristic colonies is sufficient to confirm the presence of Listeria monocytogenes . 2. If confirmation of colonies is necessary, use an API ® LIS strip or RAPIDEC ® Lmono or a Fast Rhamnose assay to directly test isolated colonies (without a purification step). In the event of discordant results (positive with the alternative method, not confirmed by one of the options described above), the laboratory must take the necessary steps to ensure that the results obtained are accurate. It is recommended, for example, to perform the following procedure: 1. Transfer 100 μL of sample from the first enrichment to 10 mL Fraser for a second enrichment. 2. Incubate at 37°C ± 1°C for 24 ± 3 hours. 3. Isolate 10 μL on chromogenic agar according to the definition of EN ISO 11290 (Ottaviani Agosti formulation type) or forming part of an ISO 16140 certified method. The presence of characteristic colonies is sufficient to confirm the presence of Listeria monocytogenes . 4. If no typical Listeria monocytogenes colony is identified, repeat steps 1 and 2, using 500 μL from the first enrichment. Confirmation of Positive Results Obtained Using AOAC RI Approved Protocols All positive results must be confirmed according to the BAM or MLG, or according to the following BIOMÉRIEUX GENE ‑ UP confirmation protocol. 9,10 Confirmation should be performed using the enrichment broth stored at +2°C/+8°C and should be initiated within 72 hours following the end of the incubation period. Using an enriched sample, mix thoroughly by hand. 1. Isolate the enrichment broth on an ALOA agar plate; incubate at +35°C ± 1°C for 48 ± 3 hours. The plate should be read after 24 and 48 hours (in case there are no typical colonies after 24 hours). 2. The presence of typical colonies confirms a positive result. 3. If identification of colonies is necessary, use an API ® LIS strip or RAPIDEC ® Lmono to directly test isolated colonies (without a purification step). In the event of discordant results (positive with the alternative method, not confirmed by one of the options described above), the laboratory must take the necessary steps to ensure that the results obtained are accurate. It is recommended, for example, to perform the following procedure: 1. Transfer 100 μL of sample from the first enrichment to 10 mL Fraser broth for a second enrichment. 2. Incubate at +35°C ± 1°C for 24 ± 3 hours. 3. Isolate on an ALOA agar plate and incubate at +35°C ± 1°C for 48 ± 3 hours. The plate should be read after 24 and 48 hours (in case there are no typical colonies after 24 hours). 4. The presence of typical colonies confirms a positive result. 5. If no typical Listeria colony is identified, repeat steps 1 and 2, using 500 μL from the first enrichment. QUALITY CONTROL External quality control can be performed using one L. monocytogenes strain. 1. Add one isolated colony from a fresh and pure culture in 10 mL of LPT broth. 2. Mix and incubate at +35°C or +37°C ± 1°C for 18 ‑ 24 hours. 3. Dilute 1/100 of the culture in LPT broth in order to obtain a suspension containing approximately 10 6 cells/mL of the strain. Follow the protocol from the Lysis section steps to Confirmation of Positive Results sections. Check that the results obtained correspond to the characteristics of the tested strains.

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