Microbiology Methods for ERP Review 3-2020

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GENE ‐ UP ® SLM 2 Collaborative Study

July 2019

i) Place the GENE - UP® PCR Tube Holder containing the PCR tubes in the plate centrifuge. j) Balance the centrifuge. Spin for 10 s. k) The plate is now ready to be processed in the GENE - UP® instrument and is stable for 2 h at 15–25°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. l) 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. k) Results and Interpretation a) 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

Salmonella spp. (640 nm)

Internal amplification Control (705 nm)

Result

+ +

+

+ +

‐ 

‐  ‐ 

+

‐ 

! Inhibition

‐ 

l) PCR Inhibition Protocol a) In case of an inhibited result, dilute the lysate to 1:3 in the control buffer: (1) Transfer 10 μ L of control buffer in an adapted microtube. (2) Follow the same procedure in the PCR PREPARATION section of this document, using 10 μ L 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. m) Confirmation of Positive Results a) All positive results must be confirmed according to the BAM, MLG, or ISO reference methods or according to the following bioMérieux GENE - UP® confirmation protocol.

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