AOAC 2019 First Action Methods

( j ) Spills should be wiped up thoroughly after treatment with bleach or a nucleic acid degradation solution. See the GENE- UP user manual for information on cleaning spills on or in the instrument. Do not autoclave solutions containing bleach. D. Sample Enrichment Allow enrichment broths to reach 15–25°C before use. In some cases, the enrichment media should be prewarmed to 37±1°C before adding to food samples. Use a blender bag containing a filter. ( a )  Milk-based PIF without probiotics, soy-based PIF (25 g). ― Add 225 mL BPW. Homogenize and incubate at 37±1°C for 18– 26 h. ( b )  Milk-based PIF with probiotics, soy-based PIF (25 g). ― Add 225 mL BPW with 10 mg/L novobiocin. Homogenize and incubate at 37±1°C for 18–26 h. ( c )  Soy ingredients (100 g). ―Add 900 mL prewarmed BPW. Homogenize and incubate at 37±1°C for 20–28 h. ( d )  Nonfat dry milk (100 g). ―Add 900 mL prewarmed BPW. Homogenize and incubate at 37±1°C for 20–28 h. ( e )  Milk-based PIF and soy-based PIF with or without probiotics (375 g). ―Add 1875 mL prewarmed BPW with 10 mg/L novobiocin. Homogenize and incubate at 37±1°C for 20–28 h. Note : To improve solubility of swelling products (for example, cereals) and for high-fat foods, follow the specific preparation techniques described in the applicate standards from the EN ISO 6887 series (1–3) and ISO 22964 standards. ( f )  Environmental samples (sponge or swab). ―Add 100 mL BPW (sponge) or 10 mL BPW (swab). Homogenize and incubate at 37±1°C for 16–24 h. Note : For environmental samples, collection device should first be dampened with a sterile diluent (e.g., BPW) 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 enriched sample; place it in a prelabeled 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 the 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 eight 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 sample into the lysis tube. Use the plate map from the GENE‑UP Routine software to pipet each sample into the correct plate position. ( f ) Remove the GENE‑UP lysis tube holder from the GENE‑UP heavy rack holder. ( g ) Clip the GENE‑UP lysis tube holder on the Troemner vortex mixer adaptor. ( h ) Run the vortex mixer at 2200 rpm for 5 min.

Note : When using the Vortex‑Genie Pulse, fit it with the GENE‑UP lysis rack adaptor. Run the vortex mixer at maximum speed for 5 min. The maximum speed must be above 2000 rpm. ( i ) When lysis is complete, remove the GENE‑UP lysis tube holder from the Troemner vortex mixer adaptor. ( j ) Clip the GENE‑UP lysis tube holder into the GENE‑UP heavy rack holder and proceed to final setup for PCR. Note : 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, put on a clean pair of powder- free latex or nitrile gloves. ( a ) Use the plate map created in the GENE‑UP Routine software to determine the number of PCR tubes required from the GENE‑UP PCR kit, and place the correct number of PCR tubes in the GENE‑UP PCR tube holder. If less than eight tubes in a strip are required, the strips can be cut apart, and only the used tubes are placed in the GENE‑UP PCR tube holder. Note : Only remove the required number of strips from the pouch, and carefully reseal the pouch after opening. ( b ) Use the following steps to remove the transportation caps from the strips: ( 1 ) Tap the strips on the bench to ensure the pellets are on the bottom of the tubes. ( 2 ) Carefully open the caps to prevent spilling the freeze‑dried pellets. ( 3 ) Visually check that the freeze‑dried pellets are present at the bottom of each tube. ( c ) Using a 10 μL Biotix filter pipet tip with a single or multichannel pipet, transfer 10 μL lysed sample (red) in the appropriate PCR tube. To determine the appropriate plate position for each sample, refer to the plate map from the GENE‑UP Routine software. Note : Do not agitate the lysate before aspirating the sample. The solid material must stay at the bottom of the tube. Visually check the tips to confirm the absence of beads and bubbles and the correct volume of lysate. For the negative control procedure, use 10 μL control buffer instead of lysed sample. ( d ) Place and seal the strip caps onto each strip tube using the GENE‑UP lysis tube remover tool. If less than eight caps are required, the caps can be cut apart, and only the used caps are placed onto the strip tubes. ( e ) Place the GENE‑UP PCR tube holder containing the PCR tubes in the plate centrifuge. ( f ) Balance the centrifuge. ( g ) Spin for 10 s. ( h ) The plate is now ready to be processed in the GENE‑UP instrument and is stable for 2 h at 15–25°C.

Table 2019.01B. Guidelines for interpretation of results Cronobacter species (640 nm) Internal amplification control (705 nm)

Result

+ + –

+ – +

+ + –

! Inhibition

–

–

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