AOAC 2019 First Action Methods

Note: For environmental samples, the collection device should first be dampened with a sterile diluent (e.g., buffered peptone water) containing, if necessary, a suitable neutralizing agent (e.g., Lecithin-Polysorbate-L-Histidine-Sodium thiosulfate mixture or Dey Engley). E. Sample Lysis (a) Following incubation, manually mix the contents of the blender bag. Optionally, a sterile technique can be used to remove 1 mL of enriched sample; place it in a pre-labeled microcentrifuge tube. Note: Do not discard the individual enriched samples until the analysis is complete and it has been confirmed that no further testing is required. Enriched samples can be stored at 2–8°C for up to 72 h before performing analysis. (b) Use the plate map created in the GENE-UP ® Routine software to determine the number of lysis tubes required from the GENE-UP ® Lysis Kit and place the correct number of lysis tubes in the GENE-UP ® Lysis Tube Holder. If less than 8 tubes in a strip are required, the strips can be cut apart. Note: Never open the lysis tubes. If a lysis tube opens or leaks, this should be considered a contamination event. (c) Clip the GENE-UP ® Lysis Tube Holder on the GENE-UP ® Heavy Rack Holder. (d) Transfer 20 μL of sample into the lysis tube. Use the Plate Map from the GENE-UP ® Routine software to pipet each sample into the correct plate position. (e) Remove the GENE-UP ® Lysis Tube Holder from the GENE-UP ® Heavy Rack Holder. (f) Clip the GENE-UP ® Lysis Tube Holder on the Troemner Vortex Mixer Adaptor. (g) Run the vortex mixer at 2200 rpm for 5 m. Note: When using the Vortex-Genie ® Pulse, fit it with the GENE-UP ® Lysis Rack Adaptor. Run the vortex at maximum speed for 5 m. The maximum speed must be above 2000 rpm. (h) When lysis is complete, remove the GENE-UP ® Lysis Tube Holder from the Troemner Vortex Mixer Adaptor. (i) Clip the GENE-UP ® Lysis Tube Holder into the GENE-UP ® Heavy Rack Holder and proceed to Final Setup for PCR. Note: The lysate can be stored for up to 3 days at 2–8°C or at -15 to -31°C for extended storage. F. PCR Preparation [Before beginning the procedure, don a clean pair of powder-free latex or nitrile gloves.]