AOAC 2019 First Action Methods

AOAC INTERNATIONAL Official Methods Board (OMB)

2019 Methods Book (Awards) First Action April 30, 2020

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resonance energy transfer (FRET) hybridization probes. These probes consist of two different short oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the reaction cycle. The first probe for the same assay is labeled at the 3 ′ end with fluorescein; the second probe is labeled at the 5 ′ end with LC Red 640. FRET occurs only after the two probes come into close proximity from hybridizing to the template DNA. The resulting fluorescent signal from the FRET interaction, which forms a real-time amplification curve, is how the amplified target is detected by the GENE-UP Thermocycler. After the PCR cycling program finishes, the PCR products are melted to determine the presence of the target DNA. The software interprets data for each sample and gives a positive, negative, or inhibited result. B. Apparatus and Reagents The following items are available from bioMérieux, Inc. (Hazelwood, MO, USA): ( a )  GENE-UP Cronobacter .―Stock No. 421920 (192 tests). Store kit at 2–25°C. ( 1 )  PCR reagent strips .―Two pouches with 12 strips of eight tubes (2×96 tubes). ( 2 )  Optical caps .―Two pouches with 12 strips of eight tubes (2×96 caps). ( 3 )  Control buffer .―One pouch with control buffer vial (850 μL). ( b )  GENE-UP Lysis Kit. ―Stock No. 414057. ( c )  GENE-UP Thermocycler .―Stock No. 414056. ( d )  Laboratory paddle blender .—For sample homogenization. ( 1 )  SMASHER ™.―Stock No. AESAP1064. ( 2 )  SMASHER XL .―Stock No. AESAP1100. ( e )  Buffered peptone water (BPW) .—( 1 )  6×225 mL .―Stock No. 42043. ( 4 )  4×3 L .―Stock No. AEB910303/4. ( 5 )  2×5 L .―Stock No. AEB910305/2. ( 6 )  Novobiocin .―Stock No. AEB184150/10. ( f )  Advanced digital heavy-duty vortex mixer .— (1) 120 V .― Stock No. 9456TAHDUSA. ( 2 )  230 V .―Stock No. 9456TAHDEUA. ( g )  GENE-UP lysis rack adaptor .―Stock No. 414570. ( h )  Sterile blender bags.—(1) Type P filter bag .―Stock No. AES400P/50G. ( 2 )  SMASHER XL 2L bag .―Stock No. 415180. ( 3 )  SMASHER XL 4L bag .―Stock No. 415181. ( i )  Plate centrifuge.—(1) MPS1000 Mini PCR plate spinner 110V .―Stock No. 419196. ( 2 )  MPS1000 Mini PCR plate spinner 230V .―Stock No. 414555. ( j )  GENE-UP PCR tube holder .―Stock No. 414573. ( k )  GENE-UP lysis tube remover .―Stock No. 414469. ( l )  GENE-UP heavy rack holder .―Stock No. 414571. ( m )  GENE-UP lysis tube holder .―Stock No. 414572. ( n )  Troemner vortex mixer adaptor .―Stock No. 421713. ( o )  10 µL Biotix filter pipet tip .―Stock No. 419194. Confirmation materials: ( p )  Enterobacter sakazakii isolation agar (ESIA ™ ) .―Stock No. AEB20010. ( 2 )  6×90 mL .―Stock Nos. 42042 and 42729. ( 3 ) 6×225 mL mini bag. ―Stock No. 42729.

AOAC Official Method 2019.01 Cronobacter species in Select Foods and Environmental Samples GENE-UP ® Cronobacter Test Method First Action 2019

[Applicable to detection of Cronobacter ( C. sakazakii , C. dublinensis , C. muytjensii , C. malonaticus , C. turicensis , and C. universalis ) in milk-based powered infant formula (PIF) with probiotics (25 and 375 g), milk-based PIF without probiotics (25 and 375 g), soy-based PIF (25 and 375 g), nonfat dry milk (100 g), soy ingredients (100 g), and stainless-steel and plastic environmental samples.] Caution: Dispose of used or unused reagents as well as any other contaminated disposable materials following procedures for infectious or potentially infectious products. It is the responsibility of each laboratory to handle waste and effluents produced according to their type and degree of hazardousness and to treat and dispose of them (or have them treated and disposed of) in accordance with any applicable regulations. Cronobacter is a Gram-negative facultative aerobic and anaerobic rod-shaped bacterium. Care must be taken when handling samples that may contain Cronobacter . Strict compliance with biosafety level 2 (BSL-2) practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious clinical materials or cultures. While BSL-2 containment is suitable for a Cronobacter , BSL‑3 practices and equipment are recommended for activities likely to produce significant aerosols or for activities involving production quantities of this particular organism. Laboratory personnel must be adequately trained to handle pathogens before being permitted to analyze samples for Cronobacter . Follow appropriate safety guidelines when handling potentially contaminated samples. Waste should be disposed of in compliance with local and national legislation. See Table 2019.01A for a summary of results of the interlaboratory study. See tables in J. AOAC Int . for detailed results of the interlaboratory study. A. Principle The GENE-UP ® Cronobacter kit is designed for use with the GENE-UP Thermocycler. The GENE-UP Cronobacter kit contains all of the necessary components for PCR, including sample-specific primers and probes and an internal amplification control. The GENE-UP Thermocycler detects fluorescence at several wavelengths (channels) to allow for multitarget detection in the same reaction vessel. The fluorescent signal from a sample is recorded in channel 640 nm, while the fluorescent signal for an internal amplification control is recorded in channel 705 nm. The software automatically interprets the results for the internal amplification control and determines the sample result based on the outcome of the control. The assay, for both the sample and the internal amplification control, utilizes dual fluorescence

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Table 2019.01A. Summary of results for detection of Cronobacter in reconstituted powdered infant formula with probiotics by GENE-UP ® Cronobacter Inoculation level Noninoculated a Low a High a Candidate presumptive positive/total No. of samples analyzed 1/120 91/120 120/120 Candidate presumptive LPOD 0.01 (0.00, 0.05) 0.75 (0.68, 0.84) 1.00 (0.97, 1.00) s r b 0.09 (0.08, 0.17) 0.43 (0.38, 0.50) 0.00 (0.00, 0.17) s L c 0.00 (0.00, 0.04) 0.01 (0.00, 0.19) 0.00 (0.00, 0.17) s R d 0.09 (0.08, 0.11) 0.43 (0.38, 0.50) 0.00 (0.00, 0.24) P value e 0.4410 0.4292 1.0000 Candidate confirmed positive/total No. of samples analyzed 0/120 91/120 120/120 Candidate confirmed LPOD 0.00 (0.00, 0.03) 0.75 (0.68, 0.84) 1.00 (0.97, 1.00) s r 0.00 (0.00, 0.17) 0.43 (0.38, 0.50) 0.00 (0.00, 0.17) s L 0.00 (0.00, 0.17) 0.01 (0.00, 0.19) 0.00 (0.00, 0.17) s R 0.00 (0.00, 0.24) 0.43 (0.38, 0.50) 0.00 (0.00, 0.24) P value 1.0000 0.4292 1.0000 Candidate presumptive positive that confirmed positive/total No. of samples analyzed 0/120 91/120 120/120 Candidate presumptive positive that confirmed LPOD 0.00 (0.00, 0.03) 0.75 (0.68, 0.84) 1.00 (0.97, 1.00) s r 0.00 (0.00, 0.17) 0.43 (0.38, 0.50) 0.00 (0.00, 0.17) s L 0.00 (0.00, 0.17) 0.01 (0.00, 0.19) 0.00 (0.00, 0.17) s R 0.00 (0.00, 0.24) 0.43 (0.38, 0.50) 0.00 (0.00, 0.24) P value 1.0000 0.4292 1.0000 Positive reference samples/total No. of samples analyzed 0/120 101/120 120/120 Reference LPOD 0.00 (0.00, 0.03) 0.84 (0.77, 0.91) 1.00 (0.97, 1.00) s r 0.00 (0.00, 0.17) 0.38 (0.33, 0.44) 0.00 (0.00, 0.17) s L 0.00 (0.00, 0.17) 0.00 (0.00, 0.13) 0.00 (0.00, 0.17) s R 0.00 (0.00, 0.24) 0.38 (0.34, 0.43) 0.00 (0.00, 0.24) P value 1.0000 0.8686 1.0000 dLPOD (candidate versus reference) f 0.00 (–0.03, 0.03) –0.08 (–0.19, 0.02) 0.00 (–0.03, 0.03) dLPOD (candidate presumptive versus candidate confirmed) f 0.01 (–0.02, 0.05) 0.00 (–0.11, 0.11) 0.00 (–0.03, 0.03) a  Results include 95% confidence intervals. b  s r =Repeatability SD. c  s L =Among-laboratory SD. d  s R =Reproducibility SD. e P value=Homogeneity test of laboratory PODs. f  A confidence interval for dLPOD that does not contain the value 0 indicates a statistically significant difference between the two methods.

( q )  Chromogenic Cronobacter isolation (CCI) agar .―Stock No. 421580. ( r )  Cronobacter Selective Broth (CSB) .―Stock No. 421569.

C. General Instructions ( a ) The GENE-UP Cronobacter kit is for professional use only with the GENE-UP Lysis Kit. ( b ) Comply with good laboratory practices. ( c ) Do not use reagents after the expiration date indicated on the label. ( d ) Visually inspect vials before testing. Do not use vials with evidence of damage, leakage, or deterioration. ( e ) Do not mix reagents (or disposables) from different lots. ( f ) Powder-free latex or nitrile gloves are recommended for all PCR steps. ( g ) The equipment and accessories should be regularly cleaned and decontaminated. ( h ) Never remove the caps from the lysis tubes. ( i ) Do not try to remove the strip tube caps once they have been sealed to the PCR strip tubes.

( s )  API ID32E .―Stock No. 20160. ( t )  Fast Crono .―Stock No. 417425.

Additional items: ( u )  Incubators .―Capable of maintaining 37±1°C. ( v )  Adjustable variable-volume pipets .―Capable of sampling and delivering 10–20 µL. ( w )  Adjustable variable-volume pipets (single or multichannel) .―Capable of sampling and delivering 0.5–10 µL. ( x )  Compatible sterile filter pipet tips .―20 µL. ( y )  Refrigerator .―Capable of maintaining 2–8°C. ( z )  Vortex-Genie Pulse .―Stock No. SI-P236 (Scientific Industries, Inc., Bohemia, NY, USA).

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( 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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( d ) If there are no typical colonies on the selective agar, proceed as follows: ( 1 ) Transfer 0.5 mL primary enrichment into 10 mL CSB broth. ( 2 ) After incubation for 18–24 h at 41.5±1°C, isolate 10 µL onto ESIA agar or CCI agar. ( 3 ) Identify the colonies using option ( a ) or ( b ) indicated above. J. QC External QC can be performed using one Cronobacter strain. ( a ) Add one isolated colony from a fresh and pure culture in 9 mL BPW. ( b ) Mix and incubate at 37±1°C for 18–24 h. ( c ) Dilute 1/100 of the culture in BPW in order to obtain a suspension containing approximately 10 6 cells/mL. ( d ) Follow the protocol from the Sample Lysis to Confirmation of Positive Results sections. ( e ) Check that the results obtained correspond to the characteristics of the tested strains. K. Limitations of the Method The GENE‑UP Cronobacter kit has been evaluated on a large number of matrices. However, given the wide variety of products and manufacturing procedures, it is recommended to check that the composition of the matrices tested does not affect the reliability of GENE‑UP results. References: (1)  ISO 6887-1:2017: Microbiology of the

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 according to appropriate biosafety procedures. ( i ) Refer to the appropriate GENE‑UP instrument user manual for instructions on how to start a run, view results, and use the 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 2019.01B . H. PCR Inhibition Protocol ( a ) In the case of an inhibited result, dilute the lysate to 1:3 in the control buffer. ( b ) Transfer 10 μL control buffer in an adapted microtube. ( c ) Follow the same procedure as in the PCR Preparation section above, using 10 μL this dilution of lysate. Note : It is recommended to retest in parallel the lysate without dilution. In the case of inhibited results at 1:3, you can dilute the lysate to 1:10. I. Confirmation of Positive Results All positive results must be confirmed according to the ISO 22964 standard or following the 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. Mix the enriched broth thoroughly by hand prior to starting the confirmation process. ( a ) Isolate 10 µL enrichment broth on ESIA or CCI agar. Incubate the agars following the instructions in the package insert. ( 1 ) Identify between one and five typical colonies using the conventional tests described in ISO 22964:2017 (including a purification step). ( 2 ) Test isolated colonies directly using a bioMérieux API strip ID32E (without a purification step). It is not necessary to perform the oxidase test. ( 3 ) Test isolated colonies directly using a bioMérieux Fast Crono confirmation test. ( b ) Perform the current ISO 22964 procedure. Transfer 100 µL enriched sample into 10 mL CSB broth and then follow the International Organization for Standardization (ISO) procedure to obtain isolated colonies (including the purification step). Refer to the latest edition of the standard EN ISO 7218 (4) for the confirmation procedures that can be used in standardized methods. ( c ) Use of any other AFNOR NF VALIDATION certified method that bases its technology on a different principle than the GENE-UP Cronobacter method and shares the same enrichment conditions as those used for the GENE-UP Cronobacter method, the full protocol for the candidate method must start from the same common step as the GENE-UP Cronobacter method. In the event of discordant results (positive with the candidate method, not confirmed by one of the options described above), the laboratory must take the necessary steps to ensure that the results obtained are accurate. For example, isolation may be performed again using a procedure that was not followed the first time. GENE‑UP Routine software. G. Results and Interpretation

Food Chain–Preparation of Test Samples, Initial Suspension and Decimal Dilutions for Microbiological Examination–Part 1: General Rules for Preparation of the Initial Suspension and Decimal Dilutions , International Organization for Standardization, Brussels, Belgium (2)  ISO 6887-4:2017: Microbiology of the Food Chain–Preparation of Test Samples, Initial Suspension and Decimal Dilutions for Microbiological Examination–Part 4: Specific Rules for the Preparation of Miscellaneous Products , International Organization for Standardization, Brussels, Belgium (3)  ISO 6887-5:2017: Microbiology of the Food Chain–Preparation of Test Samples, Initial Suspension and Decimal dilutions for Microbiological Examination–Part 5: Specific Rules for the Preparation of Milk and Milk Products , International Organization for Standardization, Brussels, Belgium (4)  ISO 7218:2007: Microbiology of Food and Animal Feeding Stuffs–General Requirements and Guidance for Microbiological Examinations , International Organization for Standards, Brussels, Belgium

J. AOAC Int . 103 , 184(2020) DOI: 10.5740/jaoacint.19-0122 Posted: July 2019 (pre-publication); January 2020 (First Action publication)

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AOAC Official Method 2019.02 Enumeration of Total Aerobic Count in Select Foods MC-Media Pad ® Rapid Aerobic Count First Action 2019

(Applicable to the enumeration of total aerobic count in raw chicken breast, raw ground pork, cream cheese, yogurt drink, parsley, vegetable juice, prawns, tuna pâté, sandwiches and pasta salad.) See Tables 2019.02A and 2019.02B for a summary of results of the collaborative study. A. Principle The MC-Media Pad ® RAC is intended to enumerate viable total aerobic bacteria in select foods in as little as 24 hours. The MC-Media Pad ® RAC uses a special medium composition that reconstitutes automatically and a unique redox indicator for detection. Once the liquid sample is inoculated onto the test pad, the sample diffuses through the whole pad by capillary action. If target organisms are present, colonies appear red on the test pad. B. Apparatus and Reagents a. MC-Media Pad ® Rapid Aerobic Count – 100 test pads (25 pads/pouch, 4 pouches/ box). Available from MilliporeSigma #132302. b. Sterile Diluent- Butterfield’s Phosphate Buffer (BPB) c. Pipettes- capable of pipetting 1,000 µL or a serological pipette d. Sterile pipette tips- capable of 1,000 µL e. Laboratory paddle-blender- Seward 400 or equivalent f. Filter Stomacher bags - Seward or equivalent g. Incubators – Capable of maintaining 35 ± 1°C and 30 ± 1°C (ISO) h. Refrigerator - capable of maintaining 2-15°C, for storing the MC-Media Pads i. Standard Colony Counter or Illuminated Magnifier j. Top-loading balance – capable of weighing 1-2000 g C. General Instructions a. Read the instruction manual carefully before use b. Storage conditions: Store the MC-Media Pads at 2-15°C. After opening the aluminum bag, unused pads should be stored in the aluminum bag sealed with tape and kept in a cool (2-15°C) environment. After opening, use all pads within 1 month. c. Do not expose unused pads to sunlight or ultraviolet light. d. Do not use a discolored or damaged pad. e. A wrinkle on the test pad should not affect detection. f. Small fragments of fabric on or around the test pad should not affect detection. g. Do not use the pads after the expiration date. The quality of an expired product is not warranted. h. The measurement range is less than 300 CFU/pad. If more than 300 CFU/pad are counted, further dilution is recommended.

i. The nature of food (high viscosity or presence of food dye) may affect test usage or results. In that case, the cause needs to be eliminated by dilution or other means. j. The used kit must be decontaminated by autoclaving or boiling and disposed of according to local regulations for waste. Safety Precautions After use, the diluents and MC-Media Pads may contain microorganisms that may be a potential biohazard. When testing is complete, follow current industry standards for the disposal of contaminated waste. Consult the Safety Data Sheet for additional information and comply with local regulations for disposal. To reduce the risks associated with bacterial infection and workplace contamination: Perform MC-Media Pad testing in a properly equipped laboratory under the control of a skilled microbiologist. D. Sample Preparation Samples are prepared depending on what reference method is being followed. Please see below for the validated reference methods the MC-Media Pad ® Rapid Aerobic Count test pad has been compared to: 1. MLG 3.02 – Place a 50 g test portion in a filtered laboratory blender bag and add 450 mL of BPB. Homogenize with a laboratory blender. 2. SMEDP Chapter 6 – Place an 11 g or 11 mL test portion into a filtered laboratory blender bag and add 99 mL of BPB. Homogenize with a laboratory blender. 3. OMA 966.23 – Parsley (50 g), Vegetable Juice (50 mL), Cooked Prawns (50 g), Tuna Pâté (50 g), Egg Salad Sandwich (50 g), Deli Pasta Salad (50 g). Place a 50 g test portion in a filtered laboratory blender bag and add 450 mL of BPB. Homogenize with a laboratory blender. 4. ISO 4833:2013 – Raw Chicken Breast (10 g), Raw Ground Pork (10 g), Cream Cheese (10 g), Yogurt Drink (10 mL), Parsley (10 g), Vegetable Juice (10 mL), Cooked Prawns (10 g), Tuna Pâté (10 g), Egg Salad Sandwich (10 g) and Deli Pasta Salad (10 g). Place a 10 g test portion in a filtered laboratory blender bag and add 90 mL of Maximum Recovery Diluent (MRD). Homogenize with a laboratory blender. For all products, if necessary, make 10-fold serial dilutions with BPB or MRD. 5. Open the aluminum bag and remove MC-Media Pad ® . 6. Lift the transparent cover film and pipette 1.0 mL of sample solution onto test pad. (It is recommended to lift the cover film diagonally for easy and secure re-sealing). 7. Close the cover film and lightly press the edges of the film to seal. 8. For preparation of samples according to the USDA and SMEDP methods, incubate the MC-Media Pad ® RAC test plate at 35 ± 1°C for 24-48 hours. 9. For preparation of samples according to the ISO method, incubate the MC-Media Pad ® RAC test plate at 30 ± 1°C for 72 ± 3 hours. 10. After incubation, count all reddish colored colonies. 11. If a large number of colonies is difficult to count, estimate by counting colonies in one grid square and multiplying by 20. If more than 10 4 microbes are grown, the entire test pad may appear stained and it may appear that no individual colonies were formed. If this occurs, dilute the sample further and re- test.

Reference: J. AOAC Int . (future issue) Posted: April 2019

Table 2019.02A. Collaborative Study Results of MC-Media Pad ® RAC vs. USDA MLG 3.02

Difference of Means d 95% LCL, UCL

Difference of Means b,c

MC-Media Pad ® RAC

USDA MLG 3.02

Mean Log 10 CFU/g

Mean Log 10 CFU/g

Matrix

Lot

N a

s R

Lot

N

s R

s r

s r

a

Raw Ground Pork (24 Hours) Raw Ground Pork (48 Hours)

Low

9 3.438 0.08 0.12 9 4.768 0.07 0.14

Low

9 9

3.423 0.16 4.754 0.09

0.18 0.16

0.015 0.014

-0.038, 0.068 -0.013, 0.042

Medium

Medium

High

9 5.313 0.12 0.17

High

9

5.322 0.08

0.16

-0.008

-0.054, 0.037

Low

9 3.465 0.09 0.13 9 4.782 0.08 0.14

Low

9 9

3.423 0.16 4.754 0.09

0.18 0.16

0.042 0.029

-0.018, 0.101 0.005, 0.052

Medium

Medium

High

9 5.354 0.11 0.15

High

9

5.322 0.08

0.16

0.032

-0.001, 0.066

a Number of laboratories that reported complete results. b Mean Log 10 Candidate method – Mean Log 10 Reference method c A 95% confidence interval for the true mean difference within +0.5 indicates equivalence of the two methods d 95% Lower and Upper Confidence Limits

s r – Repeatability Standard Deviation s R – Reproducibility Standard Deviation

Table 2019.02B. Collaborative Study Results of MC-Media Pad ® RAC vs. SMEDP

MC-Media Pad ® RAC

SMEDP

Difference of Means d 95% LCL, UCL -0.053, 0.013 0.008, 0.043

Difference of Means b,c

Mean Log 10 CFU/g 2.311 3.766

Mean Log 10 CFU/g 2.331 3.740

Matrix

Lot

N

s R

Lot

N

s R

s r

s r

a

a

Yogurt Drink (24 Hours) Yogurt Drink (48 Hours)

Low

9 9

0.29 0.08

0.29 0.12

Low

9 9

0.09 0.06

0.12 0.12

-0.020 0.026

Medium

Medium

High

9

4.266

0.10

0.16

High

9

4.266

0.09

0.15

-0.001

-0.032, 0.030

Low

9 9

2.329 3.785

0.12 0.07

0.12 0.11

Low

9 9

2.331 3.740

0.09 0.06

0.12 0.12

-0.002 0.045

-0.029, 0.025 0.028, 0.062

Medium

Medium

High

9

4.278

0.10

0.16

High

9

4.266

0.09

0.15

0.012

-0.023, 0.047

a Number of laboratories that reported complete results. b Mean Log 10 Candidate method – Mean Log 10 Reference method c A 95% confidence interval for the true mean difference within +0.5 indicates equivalence of the two methods d 95% Lower and Upper Confidence Limits s r – Repeatability Standard Deviation s R – Reproducibility

sequence of the amplified fragment during the annealing phase of the reaction cycle. The first probe for the same assay is labeled at the 3 ˊ end with fluorescein; the second probe is labeled at the 5ˊ end with LC Red 640. FRET occurs only after the two probes come in close proximity from hybridizing to the template deoxyribonucleic acid (DNA). The resulting fluorescent signal from the FRET interaction, which forms a real-time amplification curve, is how the amplified target is detected by the GENE-UP ® Thermocycler. After the PCR cycling program finishes, the PCR product(s) are melted to determine the presence of the target DNA. The software interprets data for each sample and gives a positive, negative, or inhibited result. B. Apparatus and Reagents All items are available from bioMérieux, Inc. (Hazelwood, MO, USA) unless otherwise specified. (a)  GENE-UP ® ECO 2.— REF 423108 (192 tests). Store kit at 2–25°C. ( 1 )  PCR reagent strips.— Two pouches with 12 strips of eight tubes (2 × 96 tubes). ( 2 )  Optical caps.— Two pouches with 12 strips of eight tubes (2 × 96 caps). ( 3 )  Control buffer.— One pouch with control buffer vial (850 μ L). (b)  GENE-UP ® Lysis kit.— REF 414057. (c)  GENE-UP ® Thermocycler.— REF 414056. (d)  Laboratory paddle blender.— For sample homogenization. ( 1 )  SMASHER ® .— REF AESAP1064. ( 2 )  SMASHER ® XL.— REF AESAP1100. (e)  Buffered peptone water (BPW).— ( 1 )  6 × 225 mL.— REF 42043. ( 4 )  4 × 3 L.— REF AEB910303/4. ( 5 )  2 × 5 L.— REF AEB910305/2. (6) Acriflavin.— Major laboratory supplier. (f)  Advanced digital heavy-duty vortex mixer.— ( 1 )  120 V.— REF 9456TAHDUSA. ( 2 )  230 V.— REF 9456TAHDEUA. (g)  GENE-UP ® lysis rack adaptor.— REF 414570. (h)  Sterile blender bags.— ( 1 )  Type P filter bag.— REF AES400P/50G. ( 2 )  SMASHER ® XL bag 2 L.— REF 415180. ( 3 )  SMASHER ® XL bag 4 L.— REF 415181. (i)  Plate centrifuge.— ( 1 )  MPS1000 mini PCR plate spinner 110 V.— REF 419196. ( 2 )  MPS1000 mini PCR plate spinner 230 V.— REF 414555. ( 2 )  6 × 90 mL.— REF 42042, 42729. ( 3 )  6 × 225 mL Mini bag.— REF 42729.

AOAC Official Method 2019.03 E. coli O157:H7 in Select Foods GENE-UP® E. coli O157:H7 2 (ECO 2) Kit First Action 2019

[Applicable to detection of E. coli O157:H7 in fresh raw ground beef (73% lean; 25 and 375g), fresh spinach (200g), raw milk cheese (25g), raw ground chicken (25g), raw beef trim (25 and 375g), raw ground pork (375 g), Romaine lettuce (375 g), raw sausage (25g), raw milk (25g), raw ground veal (25 and 375g), and processed water (25mL).] See Table 2019.03A for a summary of results of the interlaboratory study. Caution:  Dispose of used or unused reagents as well as any other contaminated disposable materials following procedures for infectious or potentially infectious products. It is the responsibility of each laboratory to handle waste and effluents produced according to their type and degree of hazardousness and to treat and dispose of them (or have them treated and disposed of) in accordance with any applicable regulations. E. coli O157:H7 is a Shiga toxin-producing E. coli strain. Strains of serotype O157:H7 are a demonstrated hazard to laboratory personnel. The infectious dose is estimated to be low, 10–100 organisms. Care must be taken when handling samples that may contain E. coli O157:H7. Strict compliance with biosafety level (BSL)‑2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious clinical materials or cultures. While BSL-2 containment is suitable for all other E. coli , BSL‑3 practices and equipment are recommended for activities likely to produce significant aerosols or for activities involving production quantities of this particular organism. Laboratory personnel must be adequately trained to handle pathogens before being permitted to analyze samples for E. coli . Follow appropriate safety guidelines when handling potentially contaminated samples. Waste should be disposed of in compliance with local and national legislation. A. Principle The GENE-UP ® E. coli O157:H7 2 (ECO 2) kit is designed for use with the GENE-UP ® Thermocycler. The GENE-UP ® ECO 2 kit contains all of the necessary components for PCR, including sample-specific primers and probes and an internal amplification control. The GENE-UP ® Thermocycler detects fluorescence at several wavelengths (channels) to allow for multitarget detection in the same reaction vessel. The fluorescent signal from a sample is recorded in channel 640, while the fluorescent signal for an internal amplification control is recorded in channel 705. The software automatically interprets the results for the internal amplification control and determines the sample result based on the outcome of the control. Both the assay for the sample and the internal amplification control utilize dual fluorescence resonance energy transfer (FRET) hybridization probes. These probes consist of two different short oligonucleotides that hybridize to an internal

(j)  GENE-UP ® PCR tube holder.— REF 414573. (k)  GENE-UP ® lysis tube remover.— REF 414469. (l)  GENE-UP ® heavy rack holder.— REF 414571. (m)  GENE-UP ® lysis tube holder.— REF 414572. (n)  Troemner vortex mixer adaptor.— REF 421713. (o)  Biotix filter pipet tip.— 10 µL; REF 419194.

Confirmation materials.— All items are available from bioMérieux, Inc. (a)  Cefixime-Tellurite (CT) mixture.— REF 42606. (b)  SMAC CT agar.— REF 43391. (c)  CHROMID ® O157:H7 ID-F.— REF 42605/42630. (d)  CHROMID ® EHEC agar.— REF 413093/413697. (e)  Latex O157 , ex. SLIDEX ® E. coli O157.— REF 417407. (f)  VIDAS ® ESPT.— REF 30229.

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Table 2019.03A. Summary of results for detection of E. coli O157:H7 in raw milk cheese (Comté, 34% fat, 0.8% salt) by GENE-UP® E. coli O157:H7 2 (ECO 2) a Extraction method Inoculation level Noninoculated Low High Candidate presumptive positive/total No. of samples analyzed 1/88 79/88 87/88 Candidate presumptive LPOD (CP) 0.01 (0.00, 0.06) 0.90 (0.82, 0.95) 0.99 (0.94, 1.00) s r b 0.11 (0.09, 0.20) 0.22 (0.19, 0.27) 0.11 (0.09, 0.20) s L c 0.00 (0.00, 0.05) 0.22 (0.14, 0.39) 0.00 (0.00, 0.05) s R d 0.11 (0.09, 0.13) 0.31 (0.26, 0.46) 0.11 (0.09, 0.13) P value e 0.4305 0.0000 0.4305 Candidate confirmed positive/total No. of samples analyzed 0/88 79/88 87/88 Candidate confirmed LPOD (CC) 0.00 (0.00, 0.04) 0.90 (0.82, 0.95) 0.99 (0.94, 1.00) s r 0.00 (0.00, 0.20) 0.22 (0.19, 0.27) 0.11 (0.09, 0.20) s L 0.00 (0.00, 0.20) 0.22 (0.14, 0.39) 0.00 (0.00, 0.05) s R 0.00 (0.00, 0.28) 0.31 (0.26, 0.46) 0.11 (0.09, 0.13) P value 1.0000 0.0000 0.4305 Candidate confirmed positive/total No. of samples analyzed 0/88 79/88 87/88 Candidate presumptive positive that confirmed LPOD (C) 0.00 (0.00, 0.04) 0.90 (0.82, 0.95) 0.99 (0.94, 1.00) s r 0.00 (0.00, 0.20) 0.22 (0.19, 0.27) 0.11 (0.09, 0.20) s L 0.00 (0.00, 0.20) 0.22 (0.14, 0.39) 0.00 (0.00, 0.05) s R 0.00 (0.00, 0.28) 0.31 (0.26, 0.46) 0.11 (0.09, 0.13) P value 1.0000 0.0000 0.4305 Positive reference samples/total No. of samples analyzed 0/88 55/88 72/88 Reference LPOD 0.00 (0.00, 0.04) 0.63 (0.41, 0.84) 0.82 (0.66, 0.98) s r 0.00 (0.00, 0.20) 0.39 (0.34, 0.46) 0.33 (0.28, 0.39) s L 0.00 (0.00, 0.20) 0.31 (0.19, 0.52) 0.22 (0.12, 0.42) s R 0.00 (0.00, 0.28) 0.49 (0.42, 0.53) 0.39 (0.34, 0.53) P value 1.0000 0.0000 0.0003 dLPOD (candidate vs. reference) f 0.00 (–0.04, 0.04) 0.27 (0.04, 0.49) 0.17 (0.01, 0.33) dLPOD (candidate presumptive vs. candidate confirmed) f 0.01 (–0.03, 0.06) 0.00 (–0.09, 0.09) 0.00 (–0.05, 0.05) a  Results include 95% confidence Intervals. b s r = Repeatability standard deviation. c s L = Among-laboratory standard deviation. d s R = Reproducibility standard deviation. e P value = Homogeneity test of laboratory PODs. f  A confidence interval for dLPOD that does not contain the value 0 indicates a statistically significant difference between the two methods.

Additional items.— Available from major laboratory suppliers. (a)  Incubators.— Capable of maintaining 41.5 ± 1°C or 42 ± 1°C. (b)  Adjustable, variable volume pipets.— Capable of sampling and delivering 10–20 µL. (c)  Adjustable, variable volumepipets (singleormultichannel).— Capable of sampling and delivering 0.5–10 µL. (d)  Compatible sterile, filter pipet tips.— 20 µL. (e)  Refrigerator.— Capable of maintaining 2–8°C. (f)  Vortex-Genie Pulse.— Scientific Industries, SKU: SI-P236.

C. General Instructions (a)  GENE-UP® ECO 2 kit is for professional use only with the GENE-UP® Lysis kit. (b)  Comply with Good Laboratory Practices. (c)  Do not use reagents after the expiration date indicated on the label. (d)  Visually inspect vials before testing. Do not use vials with evidence of damage, leakage, or deterioration. (e)  Do not mix reagents (or disposables) from different lots. (f)  Powder-free latex or nitrile gloves are recommended for all PCR steps.

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(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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(c)  Isolate the enrichment broth on SMAC-CT agar and CHROMID ® O157:H7 agar or CHROMID ® EHEC after performing a VIDAS ® ESP2 assay. (d)  Incubate plates for 18–24 h at 37 ± 1°C. (e)  Identify between one and five typical colonies using O157 and H7 latex test. The O157 latex assay can be performed from an isolated colony on the selective agar. In the event of discordant results (positive with the alternative method, not confirmed by one of the options described above), the laboratory must take the necessary steps to ensure that the results obtained are accurate. For example, isolation may be performed again using a procedure that was not followed the first time. J. Quality Control External quality control can be performed using one E. coli O157:H7 strain. (a)  Add one isolated colony from a fresh and pure culture in 9 mL BPW. (b)  Mix and incubate at 37 ± 1°C for 18–24 h. (c)  Dilute 1/100 of the culture in BPW in order to obtain a suspension containing approximately 10 6 cells/mL of the strain. (d)  Follow the protocol from E , Sample Lysis , to I , Confirmation of Positive Results . (e)  Check that the results obtained correspond to the characteristics of the tested strains. K. Limitations of the Method (a)  The GENE-UP ® ECO 2 kit has been evaluated on a large number of matrices. However, given the wide variety of products and manufacturing procedures, it is recommended to check that the composition of the matrices tested does not affect the reliability of GENE-UP ® results. Note: It is the responsibility of the user to perform quality control, taking into consideration the intended use of the medium, and in accordance with any applicable local regulations (frequency, number of strains, incubation temperature, etc.). Reference : J. AOAC Int . (future issue) DOI: 10.1093/jaocint/qsz016 Posted: August 2019 (pre-publication); January 31, 2020 (revision from author); April 2020 (First Action publication)

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 Table 2019.03B . 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 F , PCR Preparation , using 10 μ L of this dilution of lysate. Note: It is recommended to retest in parallel the lysate without dilution. In case of inhibited results at 1:3, dilute the lysate to 1:10. Some matrices like aromatics herbs or cocoa powders may contain inhibitory molecules. For these matrices, 1mL of enriched sample could be diluted 1:5 or 1:10 in TS 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 after mixing thoroughly by hand and should be initiated within 72h following the end of the incubation period. (a)  Isolate the enrichment broth on SMAC-CT agar and CHROMID ® O157:H7 agar with CT or CHROMID ® EHEC with CT. (b)  Isolate the enrichment broth on SMAC-CT agar and CHROMID ® O157:H7 agar or CHROMID ® EHEC after performing a magnetic immunoseparation for E. coli O157. Table 2019.03B. Interpretation of GENE-UP results E. coli O157:H7 (640 nm) Internal amplification control (705 nm) Result + + + + – + – + – – – ! Inhibition

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AOAC Official Method 2019.04 Alcohol Content in Kombucha Tea Headspace Solid-Phase Microextraction and Gas Chromatography-Mass Spectrometry First Action 2019

A. Apparatus ( a ) Analyses were done on 6890/5973N and 7890/5977C GC/MS systems (Agilent Technologies, Santa Clara, CA, USA). ( b ) The chromatographic separation in both systems was performed on a 30 m x 0.25 mm ID, 0.50 µm d f SUPELCOWAX 10 capillary column (MilliporeSigma, Bellefonte PA, USA) with helium carrier gas. ( c ) The SPME fiber used was fused silica, 100 µm polydimethylsiloxane (PDMS), 24 gauge, installed in an autosampler-style holder (MilliporeSigma, Bellefonte PA, USA). ( d ) SPME and injection were performed using an MPS autosampler with cooled agitator (Gerstel GmbH & Co. KG, Mϋlheim an der Ru hr, Germany). ( e ) Deionized water was obtained from a Barnstead Nanopure Diamond water system (ThermoFisher Scientific, Dubuque, IA, USA). ( f ) Quantitative data analysis was done using Mass Hunter Software (Agilent Technologies, Santa Clara, CA) and method validation parameters were calculated using Microsoft Excel. B. Reagents ( a ) 200 proof ethanol (99.5 % purity) was obtained from MilliporeSigma (St. Louis, MO, USA). ( b ) Anhydrous ethanol-d6 (99%) was obtained from Cambridge Isotope Laboratories (Andover, MA, USA) and MilliporeSigma (St. Louis, MO, USA). ( c ) Sodium chloride (99.5% purity), sodium phosphate-monobasic (99%), and sodium phosphate-dibasic (99%) were obtained from MilliporeSigma (St. Louis, MO, USA). ( d ) A .05 M phosphate buffer solution containing 25% w/v sodium chloride was prepared by dissolving 7 g of sodium phosphate dibasic and 25 g sodium chloride to a final volume of 1 L in water. The resulting solution was then adjusted to pH 7 with addition of sodium phosphate monobasic. ( e ) Alcohol calibration standards were prepared at concentrations of 0.10, 0.40, 0.80, 1.00, 1.50 and 2.00 percent alcohol by volume (%ABV) by direct dilution of aliquots of 200 proof ethanol

into 25 mL of water. The resulting solutions were stored under refrigeration in 22 mL vials with minimal headspace prior to use. ( f ) Internal standard solution was prepared at a concentration of 0.08 %ABV by direct dilution of neat ethanol-d6 into the salt/buffer solution. This solution was then used in the dilution of samples prior to SPME. ( g ) Internal standard/buffer solution was prepared daily, and chilled prior to use. C. Samples ( a ) All Kombucha samples used were purchased at local grocery stores, and kept under refrigeration until testing. ( b ) A ginger flavored Kombucha found to have very low alcohol content was used for the preparation of spikes in the method validation process. ( c ) Certified reference materials of alcohol in water at 80, 200, and 400 mg/dL were obtained from Cerilliant (Roundrock, TX , USA). ( d ) IRMM certified reference materia ls of low alcohol beer, nominal 0.50 % ABV (BCR -651), were obtained from MilliporeSigma. Two separate samples of this reference material were obtained approximately eight months apart. ( e ) Calibration standards for HS-SPME were prepared by dilution of 400 µL of the alcohol calibration solutions in water with 3.6 mL of the salt/buffer solution containing internal standard at .08% ABV. This resulted in a 10X dilution of each standard solution, and a final internal standard concentration of .072 %ABV in each calibration sample. D. HS-SPME Procedure Samples for HS-SPME were prepared by dilution of 400 µL of sample with 3.6 mL of salt/buffer solution containing .08 %ABV internal standard (see reagent section) in a 10 mL headspace vial. This results in a 10X dilution of the sample and a final internal standard concentration of .072 %ABV. Prior to extraction, samples were incubated at 40°C for 7 min in a heated agitator with agitation speed at 250 rpm. Extraction was performed by exposing a 100 µm PDMS SPME fiber to the sample headspace for 2 min at 40°C at the same agitation speed. After extraction, the fiber was desorbed in the GC inlet for 3 min at 250°C. A post-bake of the SPME fiber was done after each extraction for 5 min at 260 °C. E. Chromatographic Conditions ( a ) The GC oven temperature was programmed at 40 ° C for 5 min, ramped at 8 °C/min to 70°C, ramped at 20°C/min to 250°C and held for 5 min.

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