AOAC 2019 First Action Methods

(g)  Equipment and accessories should be regularly cleaned and decontaminated. (h)  Never remove caps from lysis tubes. (i)  Do not try to remove strip tube caps once they have been sealed to the PCR strip tubes. (j)  Spills should be wiped up thoroughly after treatment with bleach or a nucleic acid degradation solution. See 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, enrichment media should be prewarmed to 41.5 ± 1°C or 42±1°C before adding to food samples. Frozen samples should be thawed before analysis. To decrease occurrence of stressed cells, use a quick-thawing process. (This can be performed by thawing at temperatures less than 45°C in a water bath for no more than 15min. Agitate the sample continuously.) Use a blender bag containing filter. (a)  Fresh raw ground beef (25 g). —Add 225 mL BPW. Homogenize and incubate at 42 ± 1°C for 18–24 h. (b)  Fresh raw ground beef: short protocol (25 g). —Add 225 mL prewarmed BPW. Homogenize and incubate at 42 ± 1°C for 8–24 h. (c)  Raw ground chicken (25 g). —Add 225 mL prewarmed BPW or modified tryptic soy broth (mTSB). Homogenize and incubate at 42 ± 1°C for 18–24 h. (d)  Fresh spinach (200 g). —Add 800 mL BPW. Homogenize and incubate at 42 ± 1°C for 18–24 h. (e)  Raw beef trim (325 g). —Add 975 mL mTSB. Homogenize and incubate at 42 ± 1°C for 15–24 h. (f)  Raw beef trim, raw ground pork, and raw ground beef (375 g). —Add 1125 mL prewarmed (42 ± 1°C) BPW or mTSB. Homogenize and incubate at 42 ± 1°C for 10–24 h. mTSB enrichment is applicable to raw ground pork. (g)  Romaine lettuce (375 g). —Add 1125 mL prewarmed BPW. Homogenize and incubate at 41.5 ± 1°C for 22–24 h. E. Sample Lysis (a)  Following incubation, manually mix contents of the blender bag. Optionally, a sterile technique can be used to remove 1 mL enriched sample; place in a prelabeled microcentrifuge tube. Note: Do not discard the individual enriched samples until 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 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 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 min. Note: When using the Vortex-Genie ® Pulse, fit it with the GENE- UP ® lysis rack adaptor. Run the vortex at maximum speed for 5 min. Maximum speed must be >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: Lysate can be stored for up to 3days 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. (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 transportation caps from the strips: ( 1 ) Tap the strips on the bench to ensure pellets are on the bottom of the tubes. ( 2 ) Carefully open the caps to prevent spilling the freeze‑dried pellet. ( 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 μ L of 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, bubbles, and for correct volume of lysate. For negative control procedure, use 10 μ L 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. Note: 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 according to appropriate biosafety procedures. (i)  Refer to appropriate GENE‑UP ® instrument user manual for instructions to start a run, view results, and use the GENE‑UP ® Routine software.

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