ERP Micro December 2019

GENE-UP ® Listeria spp. 2 (LIS 2)

050567 - 02 - 2018-12 - en

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 on 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. 8. The plate is now ready to be processed in the GENE ‑ UP ® instrument and is stable for 2 hours at +15°C/+25°C. 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. 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. 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.

Internal amplification control (705 nm)

Listeria spp. (640 nm)

Result

+ +

+

+ +

-

- -

+

-

-

! 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 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.

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