ERP Micro December 2019

GENE-UP ® L. monocytogenes 2 (LMO 2)

050568 - 02 - 2018-12 - en

6. Run the vortex mixer at 2200 rpm for 5 minutes. Note: When using the Vortex ‑ Genie ® Pulse, fit it with the GENE ‑ UP ® Lysis Rack Adaptor. Run the vortex at maximum speed for 5 minutes. The maximum speed must be above 2000 rpm. 7. When lysis is complete, remove the GENE ‑ UP ® Lysis Tube Holder from the Troemner Vortex Mixer Adaptor. 8. Clip the GENE ‑ UP ® Lysis Tube Holder into the GENE ‑ UP ® Heavy Rack Holder, and proceed to Final Setup for PCR. The lysate can be stored for up to 3 days at +2°C/+8°C or at -15°C/-31°C for extended storage. FINAL SETUP FOR PCR Before beginning the procedure, put on a clean pair of powder ‑ free latex or nitrile gloves. 1. Use the plate map created in the GENE ‑ UP ® Routine software to determine the number of PCR tubes required from the GENE ‑ UP ® PCR Kit and place the correct number of PCR tubes in the GENE ‑ UP ® PCR Tube Holder. If less than eight tubes in a strip are required, the strips can be cut apart, and only the used tubes are placed in the GENE ‑ UP ® PCR Tube Holder. Note: Only remove the required number of strips from the pouch and carefully reseal the pouch after opening. 2. Use the following steps to remove the transportation caps from the strips: a. Tap on the strips on the bench to ensure the pellets are at the bottom of the tubes. 3. Using a 10 μL Biotix filter pipette tip with a single or multichannel pipette, transfer 10 μL of lysed sample (red) in the appropriate PCR tube. To determine the appropriate plate position for each sample, refer to the Plate Map from the GENE ‑ UP ® Routine software. Note: Do NOT agitate the lysate before aspirating the sample. The solid material must stay at the bottom of the tube. Note: Visually check the tips to confirm the absence of beads, bubbles, and for correct volume of lysate. Note: Check that the sample becomes purple when added to the freeze ‑ dried pellet. If the sample does not turn purple, discard the well and restart the procedure. Note: For negative control procedure, use 10 μL of control buffer instead of lysed sample. 4. Place and seal the strip caps onto each strip tube using the GENE ‑ UP ® Lysis Tube Remover Tool. If less than eight caps are required, the caps can be cut apart, and only the used caps are placed onto the strip tubes. 5. Place the GENE ‑ UP ® PCR Tube Holder containing the PCR tubes in the plate centrifuge. 6. Balance the centrifuge. 7. Spin for 10 seconds. Note: The lysis tubes can be removed from the GENE ‑ UP ® Lysis Tube Holder using the GENE ‑ UP ® Lysis Tube Remover Tool. The GENE ‑ UP ® Lysis Tube Holder is reusable, but the used lysis tubes should be disposed of accordingly. STARTING A RUN AND DISPLAYING RESULTS Please refer to the appropriate GENE ‑ UP ® instrument User Manual for instructions to start a run, view results, and use the GENE ‑ UP ® Routine software. Note: For protocols validated in the scope of NF VALIDATION, the software version is indicated in the certificate BIO ‑ 12/40 ‑ 11/16. RESULTS AND INTERPRETATION Results are automatically interpreted once the PCR run is completed. The Routine software interprets data for each sample and gives a positive, negative, or inhibited result as indicated in the following table. L. monocytogenes spp. (640 nm) Internal amplification control (705 nm) Result + + + + - + - + - - - ! inhibition 8. The plate is now ready to be processed in the GENE ‑ UP ® instrument. Note: If necessary, the PCR mix is stable for 2 hours at +15°C/+25°C. b. Carefully open the caps to prevent spilling the freeze ‑ dried pellet. c. Visually check the bottom of each tube for the freeze ‑ dried pellets.

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.

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