AOAC 2019 First Action Methods

G.

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 Table 2019.11B . 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: 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. The plate should be read after 24 and 48 hours (in case there are no typical colonies after 24 h). (b) The presence of typical colonies confirms a positive result. (c) If identification of colonies is necessary, use an API ® LIS strip or RAPIDEC ® Lmono to directly test isolated colonies (without a purification step). In the event of discordant results such as a positive result with the GENE-UP ® test, or no confirmation using a plate (no typical colonies), 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: (d) Transfer 100 μL from the primary enrichment to 10 mL FB for a secondary enrichment. Incubate at 35 ± 1°C for 24 ± 3 h. 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.