ERP Micro December 2019

Internal amplification Control (705 nm)

Listeria monocytogenes (640 nm)

Result

+ +

+

+ +

-

- -

+

-

! Inhibition

-

H. PCR Inhibition Protocol

(a) In case of an inhibited result, dilute the lysate to 1:3 in the control buffer: (b) Transfer 10 μL of control buffer in an adapted microtube. (c) Follow the same procedure in the PCR PREPARATION 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.

I. Confirmation of Positive Results

[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.] [Confirmation should be performed using the enrichment broth stored at 2–8°C and should be initiated within 72 h following the end of the incubation period. If using an enriched sample, mix thoroughly by hand.] (a) Isolate the enrichment broth on an ALOA agar plate; incubate at 35 ± 1°C for 48 ± 3 h.

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