Microbiology Methods for ERP Review 3-2020

DRAFT 1 - AOAC - 05DEC2018

156

GENE-UP ® Salmonella 2 (SLM 2)

050555 - 02 - 2018-12 - en

Salmonella spp. (640 nm)

Internal amplification control (705 nm)

Result

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! inhibition

PCR INHIBITION PROTOCOL In case of an inhibited result, dilute the lysate to 1:3 in the control buffer: 1. Transfer 10 μL of control buffer in an adapted microtube. 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 like aromatics herbs or cocoa powders may contain inhibitory molecules. For these matrices, 1 mL of enriched sample could be diluted 1:5 or 1:10 in Tryptone Salt or Normal Saline prior to the lysis step. If inhibition persists, proceed with an additional 1:3 dilution as described above. CONFIRMATION OF POSITIVE RESULTS Confirmation of Positive Results Obtained Using the Method Certified NF VALIDATION and Outside NF VALIDATION or AOAC RI Certification Protocols Confirmation should be performed using the enrichment broth stored at 2 ‑ 8°C and should be initiated within 72 hours following the end of the incubation period (except for the pet food samples specific protocol 375 g). If using an enriched sample, mix thoroughly by hand. Note: For short protocols, the confirmation can be made at the end of the incubation time (8 hours or 10 hours). 1. Isolate 10 μL of the enrichment broth using a loop onto an ASAP ™ , CHROMID ® Salmonella , or XLD plate. 2. Incubate the agar following the instructions in the package insert. Then perform one of the following procedures: a. Identify between one and five typical colonies using the conventional tests described in the methods standardized by the CEN or ISO (including the purification step). b. Test isolated colonies directly using a bioMérieux API ® strip (without a purification step). It is not necessary to perform the oxidase test. c. If a CHROMID ® Salmonella or an ASAP ™ agar is used, or if a new isolation is performed on Trypticase Soy agar, test isolated colonies directly using the Salmonella spp. latex kit (follow the instructions in the instructions for use). 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. If there are no typical colonies on the selective agar: a. Transfer 0.5 mL of enrichment broth into 10 mL of SX2 broth. b. After incubation for 6 ‑ 24 hours at 41.5°C ± 1°C, isolate 10 μL onto ASAP ™ , CHROMID ® Salmonella , or XLD. c. Identify the colonies using one of the three options indicated above. 2. If the latex test is negative, perform a confirmatory test based on a different principle (example: a BIOMÉRIEUX API ® 20E strip directly using isolated colonies, without a purification step. It is not necessary to perform the oxidase test). 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. 8,9 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. If using an enriched sample, mix thoroughly by hand. Note: For short protocols, the confirmation can be made at the end of the incubation time (8 hours or 10 hours). 1. Isolate the enrichment broth on a SALSA ™ agar plate or on ASAP ™ and XLD agar plates; incubate at 35°C ± 1°C for 24 ± 3 hours. 2. If a typical colony is obtained, test an isolated colony directly using a Salmonella spp. latex or API ® 20E strip. 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:

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