AOAC 2019 First Action Methods

( h ) 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. ( i ) 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. G. 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 Table 2019.10B . 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 ho urs (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 to directly test isolated colonies (without a purification step). 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.