AOAC 2018 Methods

AOAC INTERNATIONAL Official Methods Board (OMB)

2018 Methods Book (Awards)

April 30, 2020

AOAC INTERNATIONAL 2275 Research Blvd., Suite 300 Rockville, MD, 20850 UNITED STATES

dboyd@aoac.org 301.924.7077 x126

AOAC Official Method 2018.01 Cronobacter species in Select Foods and Environmental Surfaces 3M ™ Molecular Detection Assay (MDA) 2 – Cronobacter Method First Action 2018

C. General Instructions ( a ) Store the 3MMDA2 – Cronobacter at 2–8°C. Do not freeze. Keep kit away from light during storage. After opening kit, check that foil pouch is undamaged. If pouch is damaged, do not use. After opening, unused reagent tubes should always be stored in the resealable pouch with the desiccant inside to maintain stability of lyophilized reagents. Store resealed pouches at 2–8°C for no longer than 60 days. Do not use 3MMDA 2 – Cronobacter past expiration date. ( b ) Follow all instructions carefully. Failure to do so may lead to inaccurate results. ( c ) The 3M MDA 2 – Cronobacter is intended for use in a laboratory environment by professionals trained in laboratory techniques. 3M has not documented the use of this product in industries other than the food and beverage industries. For example, 3M has not documented this product for testing drinking water, pharmaceutical, cosmetics, clinical, or veterinary samples. The 3M MDA 2 – Cronobacter has not been evaluated with all possible food products, food processes, testing protocols, or with all possible strains of bacteria. ( d ) As with all test methods, the source of enrichment medium can influence results. The 3M MDA 2 – Cronobacter has only been evaluated for use with the enrichment media specified in the Instructions for Use section. D. Safety Precautions The 3M Molecular Detection Instrument is intended for use with samples that have undergone heat treatment during the assay lysis step, which is designed to destroy organisms present in the sample. Samples that have not been properly heat treated during the assay lysis step may be considered a potential biohazard and should not be inserted into the 3M MDS Instrument. The user should not exceed the recommended temperature setting on the heater or the recommended heating time. Use an appropriate, calibrated thermometer to verify the 3M Molecular Detection Heat Block Insert temperature (e.g., a partial immersion thermometer or digital thermocouple thermometer, not a total immersion thermometer). The thermometer must be placed in the designated location in the 3M Molecular Detection Heat Block Insert. The user should read, understand, and follow all safety information in the instructions for the 3M MDS and the 3M MDA 2 – Cronobacter . Retain the safety instructions for future reference. To reduce the risks associated with exposure to chemicals and biohazards, perform pathogen testing in a properly equipped laboratory under the control of trained personnel. Always follow standard laboratory safety practices, including wearing appropriate protective apparel and eye protection while handling reagents and contaminated samples. Avoid contact with the contents of the enrichment media and reagent tubes after amplification. Dispose of enriched samples according to current industry standards. After use, the enrichment medium and the 3M MDA 2 –  Cronobacter tubes can potentially contain pathogenic materials. Periodically decontaminate laboratory benches and equipment (pipets, cap/decap tools, etc.) with a 1–5% (v/v in water) household bleach solution (5250–6500 ppm) or DNA removal solution. When testing is complete, follow current industry standards for disposal of contaminated waste. Consult Safety Data Sheet for additional information and local regulations for disposal.

[Applicable to detection of Cronobacter species in powdered infant formula with probiotics (10 and 300 g), powdered infant cereal without probiotics (10 and 300 g), lactose powder (10 g), and environmental surface sponges (stainless steel).] See Table 2018.01 for a summary of results of the collaborative study. See additional table in J. AOAC Int . 102 , 108(2019) for detailed results of the collaborative study. A. Principle The 3M MDA 2 – Cronobacter method is used with the 3M MDS for the rapid and specific detection of Cronobacter species in select enriched food and food process environmental samples. The 3M MDA 2 – Cronobacter uses loop-mediated isothermal amplification (LAMP) of unique DNA target sequences with high specificity and sensitivity combined with bioluminescence to detect the amplification. Presumptive positive results are reported in real-time, and negative results are displayed after the assay is completed. Samples are pre-enriched in buffered peptone water (BPW)-ISO formulation. B. Apparatus and Reagents Items B(b) –( g) are available as the 3MMDA2 – Cronobacter kit from 3M Food Safety (St. Paul, MN, USA). Items B(a) –( n) and ( y ) are available from 3M Food Safety. ( a )  3M Molecular Detection System (MDS100) . ( b )  3M Molecular Detection Assay 2 – Cronobacter reagent tubes .—Twelve strips of eight tubes. ( c )  Lysis solution (LS) tubes .—Twelve strips of eight tubes. ( j )  3M Molecular Detection Cap/Decap Tool for reagent tubes. ( k )  3M Molecular Detection Cap/Decap Tool for lysis tubes. ( l )  Empty lysis tube rack . ( m )  Empty reagent tube rack . ( n )  BPW-ISO formulation . ( o )  Micropipet .—Capable of 20 μL. ( p )  Multichannel (8-channel) pipet .—Capable of 20 μL. ( q )  Sterile filter pipet tips —Capable of 20 μL. ( r )  Filter Stomacher ® bags .—Seward or equivalent. ( s )  Stomacher .—Seward or equivalent. ( t )  Thermometer .—Calibrated range to include 100 ± 1°C. ( u )  Dry block heater unit .—Capable of maintaining 100 ± 1°C. ( v )  Incubators .—Capable of maintaining 37 ± 1°C. ( w )  Refrigerator .—Capable of maintaining 2–8°C for storing the 3M MDA components. ( x )  Computer .—Compatible with the 3M Molecular Detection Instrument. ( y )  3M Hydrated Sponge Stick . ( d )  Extra caps .—Twelve strips of eight caps. ( e )  Reagent control (RC) .—Eight reagent tubes. ( f )  Quick Start Guide. ( g )  3M Molecular Detection Speed Loader Tray. ( h )  3M Molecular Detection Chill Block Insert. ( i )  3M Molecular Detection Heat Block Insert.

© 2019 AOAC INTERNATIONAL

Table 2018.01. Summary of results for detection of Cronobacter in powdered infant formula with probiotics Method a Inoculation level 3M MDA 2 – Cronobacter Uninoculated Low

High

Candidate presumptive positive/total No. of samples analyzed

2/168

88/168

168/168

LPOD CP

0.01 (0.01, 0.04) 0.11 (0.10, 0.15) 0.00 (0.00, 0.04) 0.11 (0.10, 0.12)

0.52 (0.45, 0.60) 0.51 (0.46, 0.52) 0.00 (0.00, 0.15) 0.51 (0.46, 0.52)

1.00 (0.98, 1.00) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

s r s L s R

P value b

0.5158

0.7750 86/168

1.0000 168/168

Candidate confirmed positive/total No. of samples analyzed

0/168

LPOD CP

0.00 (0.00, 0.02) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

0.51 (0.43, 0.59) 0.51 (0.46, 0.52) 0.00 (0.00, 0.11) 0.51 (0.47, 0.52)

1.00 (0.98, 1.00) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

s r s L s R

P value

1.0000

0.9494 84/168

1.0000 168/168

Candidate confirmed positive/total No. of samples analyzed Candidate presumptive positive that confirmed LPOD C

0/168

0.00 (0.00, 0.02) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

0.50 (0.42, 0.58) 0.51 (0.46, 0.52) 0.00 (0.00, 0.13) 0.51 (0.47, 0.52)

1.00 (0.98, 1.00) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

s r s L s R

P value

1.0000

0.9462 86/168

1.0000 168/168

Positive ref. samples/total No. of samples analyzed

0/168

Ref. LPOD

0.00 (0.00, 0.02) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

0.51 (0.43, 0.59) 0.51 (0.46, 0.52) 0.00 (0.00, 0.11) 0.51 (0.47, 0.52)

1.00 (0.98, 1.00) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

s r s L s R

P Value

1.0000

0.9494

1.0000

dLPOD (candidate vs. ref.) c

0.00 (–0.02, 0.02) 0.01 (–0.01, 0.04)

–0.01 (–0.12, 0.10) 0.01 (–0.10, 0.12)

0.00 (–0.02, 0.02) 0.00 (–0.02, 0.02)

dLPOD (candidate presumptive vs. candidate confirmed) c

a  Results include 95% confidence intervals. b P value = Homogeneity test of laboratory PODs. c  A confidence interval for dLPOD that does not contain the value 0 indicates a statistically significant difference between the two methods.

To reduce the risks associated with environmental contamination, follow current industry standards for disposal of contaminated waste. E. Sample Enrichment Food matrixes.— ( a ) Allow BPW-ISO to equilibrate to ambient laboratory temperature (20–25°C) for 10 g test portions or environmental samples or to 37°C for 300 g test portions. ( b ) Enrich samples following a 1:9 enrichment ratio. ( 1 ) For example, to 10 g test portions, a 90 mL volume of BPW- ISO is added. ( 2 ) For 300 g powdered infant formula and powdered infant cereal with probiotics, 10 mg/L Vancomycin is required to be supplemented into 2700 mL BPW-ISO. ( c ) Homogenize thoroughly by blending, stomaching, vortex mixing, or hand mixing for 2 ± 0.2 min, or until all lumps are completely dissolved and the enrichment suspension is homogeneous. ( d )  Incubation .—( 1 ) Incubate powdered infant formula and powdered infant cereal (10 g) for 18–20 h at 37 ±1°C.

( 2 ) Incubate powdered infant formula nonprobiotic (300 g) for 18–24 h at 37 ± 1°C. ( 3 ) Incubate powdered infant formula and powdered infant cereal with probiotics (300 g) for 22–24 h at 37 ± 1°C. ( 4 ) Incubate lactose (10 g) for 18–24 h at 37 ±1°C. Environmental samples .— ( a ) Sample collection devices should be a sponge-hydrated with Dey-Engley Neutralizing Broth. It is recommended to sanitize the area after sampling. ( b ) The recommended size of the sampling area to verify the presence or absence of the pathogen on the surface is at least 100 cm 2 (10 × 10 cm or 4 × 4 in.). When sampling with a sponge, cover the entire area going in two directions (left to right then up and down) or collect environmental samples following current sampling protocol or ISO 18593:2004 guidelines. ( c ) Allow BPW-ISO to equilibrate to ambient laboratory temperature (20–25°C). ( d ) Enrich samples by adding a 90 mL volume BPW-ISO to a sampling sponge. ( e ) Homogenize thoroughly by stomaching or hand mixing for 2 ± 0.2 min. Incubate at 37 ± 1°C for 18–24 h.

© 2019 AOAC INTERNATIONAL

Figure 2018.01A.

on the laboratory bench for at least 2 h, incubate the LS tubes in a 37 ± 1°C incubator for 1 h, or place them in a dry double block heater for 30 s at 100 ± 1°C. ( b ) Invert the capped tubes to mix. Proceed to the next step within 4 h after inverting. ( c ) Remove the enrichment broth from the incubator. ( d ) One LS tube is required for each sample and the negative control (NC; sterile enrichment medium) sample. (1) LS tube strips can be cut to desired LS tube number. —Select the number of individual LS tubes or 8-tube strips needed. Place the LS tubes in an empty rack. ( 2 ) To avoid cross-contamination, decap one LS tube strip at a time and use a new pipet tip for each transfer step. ( 3 ) Transfer enriched sample to LS tubes. Transfer each enriched sample into individual LS tube first. Transfer the NC last. ( 4 ) Use the 3M Molecular Detection Cap/Decap Tool-Lysis to decap one LS tube strip one strip at a time. ( 5 ) Discard the LS tube cap. If lysate will be retained for retest, place the caps into a clean container for reapplication after lysis. ( 6 ) Agitate the enrichment bag before collecting the sample from the filtered side when working with viscous samples. ( 7 ) Transfer 20 μL sample into a LS tube ( see Figure 2018.01A ). ( e ) Repeat steps ( 1 )–( 4 ) as needed for the number of samples to be tested. When all samples have been transferred, then transfer 20 μL NC into a LS tube. Do not recap tubes. ( f ) Verify that the temperature of the 3M Molecular Detection Heat Block Insert is at 100 ± 1°C. Place the rack of LS tubes in the 3MMolecular Detection Heat Block Insert and heat for 15 ± 1 min. During heating, the LS solution will change from pink (cool) to yellow (hot). Samples that have not been properly heat treated during the assay lysis step may be considered a potential biohazard and should not be inserted into the 3M Molecular Detection Instrument ( g ) Remove the uncovered rack of LS tubes from the heating block and allow to cool in the 3M Molecular Detection Chill Block Insert at least 5 min and a maximum of 10 min. The 3M Molecular Chill Block Insert, used at ambient temperature (20–25°C) without the Molecular Detection Chill Block Tray, should sit directly on the laboratory bench. When cool, the lysis solution will revert to a pink color. ( h ) Remove the rack of LS tubes from the 3M Molecular Detection Chill Block Insert. K. Amplification ( a ) One 3M MDA 2 – Cronobacter reagent tube is required for each sample and the NC. ( 1 ) Reagent tubes strips can be cut to desired tube number. Select the number of individual reagent tubes or 8-tube strips needed. ( 2 ) Place reagent tubes in an empty rack.

F. Preparation of the 3M Molecular Detection Speed Loader Tray ( a ) Wet a cloth or paper towel with a 1–5% (v/v in water) household bleach (5250–6500 ppm) solution and wipe the 3M Molecular Detection Speed Loader Tray. ( b ) Rinse the 3M Molecular Detection Speed Loader Tray with water. ( c ) Use a disposable towel to wipe the 3M Molecular Detection Speed Loader Tray dry. ( d ) Ensure the 3M Molecular Detection Speed Loader Tray is dry before use. G. Preparation of the 3M Molecular Detection Chill Block Insert Place the 3M Molecular Detection Chill Block Insert directly on the laboratory bench; the 3M Molecular Detection Chill Block Tray is not used. Use the block at ambient laboratory temperature (20–25°C). H. Preparation of the 3M Molecular Detection Heat Block Insert Place the 3M Molecular Detection Heat Block Insert in a dry double block heater unit. Turn on the dry block heater unit and set the temperature to allow the 3M Molecular Detection Heat Block Insert to reach and maintain a temperature of 100 ± 1°C. Note: Depending on the heater unit, allow approximately 30 min for the 3M Molecular Detection Heat Block Insert to reach temperature. Using an appropriate, calibrated thermometer (e.g., a partial immersion thermometer, digital thermocouple thermometer, not a total immersion thermometer) placed in the designated location, verify that the 3M Molecular Detection Heat Block Insert is at 100 ± 1°C. I. Preparation of the 3M Molecular Detection Instrument ( a ) Launch the 3M Molecular Detection Software and log in. Contact your 3M Food Safety representative to ensure you have the most updated version of the software. ( b ) Turn on the 3M Molecular Detection Instrument. ( c ) Create or edit a run with data for each sample. Refer to the 3M MDS User Manual for details. Note: The 3M Molecular Detection Instrument must reach and maintain temperature of 60°C before inserting the 3M Molecular Detection Speed Loader Tray with reaction tubes. This heating step takes approximately 20 min and is indicated by an ORANGE light on the instrument’s status bar. When the instrument is ready to start a run, the status bar will turn GREEN. J. Lysis ( a ) Allow the LS tubes to warm up by setting the rack at room temperature (20–25°C) overnight (16–18 h). Alternatives to equilibrate the LS tubes to room temperature are to set the LS tubes

© 2019 AOAC INTERNATIONAL

( 3 ) Avoid disturbing the reagent pellets from the bottom of the tubes. ( b ) Select one RC tube and place in rack. ( c ) To avoid cross-contamination, decap one reagent tubes strip at a time and use a new pipet tip for each transfer step. ( d ) Transfer lysate to reagent tubes and RC tube. Transfer each sample lysate into individual reagent tubes first followed by the NC. Hydrate the RC tube last. ( e ) Use the 3M Molecular Detection Cap/Decap Tool-Reagent to decap the reagent tubes one reagent tubes strip at a time. Discard cap. ( 1 ) Transfer 20 μL sample lysate from the upper half of the liquid (avoid precipitate) in the LS tube into corresponding reagent tube. Dispense at an angle to avoid disturbing the pellets. Mix by gently pipetting up and down five times. ( 2 ) Repeat step I(e) ( 1 ) until each individual sample lysate has been added to a corresponding reagent tube in the strip. ( 3 ) Cover the reagent tubes with the provided extra cap and use the rounded side of the 3M Molecular Detection Cap/Decap Tool- Reagent to apply pressure in a back and forth motion ensuring that the cap is tightly applied. ( 4 ) Repeat I(e) ( 1 ) – ( 3 ) as needed for the number of samples to be tested. ( 5 ) When all sample lysates have been transferred, repeat I(e) ( 1 ) – ( 3 ) to transfer 20 μL NC lysate into a reagent tube. ( 6 ) Transfer 20 μL NC lysate into an RC tube. Dispense at an angle to avoid disturbing the pellets. Mix by gently pipetting up and down five times. ( f ) Load capped tubes into a clean and decontaminated 3M Molecular Detection Speed Loader Tray. See Figure 2018.01B . Close and latch the 3M Molecular Detection Speed Loader Tray lid. ( g ) Review and confirm the configured run in the 3M Molecular Detection Software.

Figure 2018.01B.

( h ) Click the “Start” button in the software and select instrument for use. The selected instrument’s lid automatically opens. ( i ) Place the 3M Molecular Detection Speed Loader Tray into the 3M MDS Instrument and close the lid to start the assay. Results are provided within 60 min, although positives may be detected sooner. ( j ) After the assay is complete, remove the 3M Molecular Detection Speed Loader Tray from the 3M Molecular Detection Instrument and dispose of the tubes by soaking in a 1–5% (v/v in water) household bleach (5250–6500 ppm) solution for 1 h and away from the assay preparation area. Note: To minimize the risk of false positives from cross- contamination, never open reagent tubes containing amplified DNA. This includes RC, reagent, and matrix control tubes. Always dispose of sealed reagent tubes by soaking in a 1–5% (v/v in water) household bleach (5250–6500 ppm) solution for 1 h and away from the assay preparation area. Reference: J. AOAC Int . 102, 108(2019) DOI: https://doi.org/10.5740/jaoacint.18-0233 Posted: October 2018, February 2019

© 2019 AOAC INTERNATIONAL

108  B ird et al . : J ournal of AOAC I nternational V ol . 102, N o . 1, 2019

Food Biological Contaminants

Evaluation of the 3M™ Molecular Detection Assay (MDA) 2 – Cronobacter for the Detection of Cronobacter species in Select Foods and Environmental Surfaces: Collaborative Study, First Action 2018.01 P atrick B ird , M. J oseph B enzinger J r ., B en B astin , E rin C rowley , J ames A gin , and D avid G oins Q Laboratories, Inc., 1400 Harrison Ave, Cincinnati, OH 45214 L isa M onteroso 1 3M Food Safety Department, 3M Center, Building 260-6B-01, St. Paul, MN 55144 Collaborators: T. Abramovic; M. Achen; E. Budge; N. Clemens; A. Cruz; J. Cuellar; C. Diaz; Y. Hong; S. Ishii; C. Liska; C. Luquin; L. Ma; K. Martin; G. Masanz; M. Matijevic; P. McKelvey; J. Miller; S. Molina; A. Mustafic; I. Podoreski; I. Ricki; B. Schindler; A. Scollon; C. Somoza; C. Tinajero; L. Thompson; S. Wheeler; A. Winslow; W. Xu

formula (2). The organism is able to survive in low-moisture products, such as powdered infant formula, for long periods of time. Current manufacturing processes for producing powdered infant formula are not effective in eliminating the bacteria completely from the product (3). The addition of probiotics to these products, although beneficial for the recipient, can make it difficult to culture the pathogen due to background flora and bacteriocins produced by the probiotic culture. The 3M ™ Molecular DetectionAssay (MDA) 2 – Cronobacter method, uses loop-mediated isothermal amplification (LAMP) for deoxyribonucleic acid (DNA) amplification of the target organism in real-time. This involves a two-step enzymatic process in which pyrophosphate molecules, produced as a byproduct of the DNA amplification, are used to generate light. This light emission is measured by the 3M Molecular Detection Instrument and signals the detection of the target organism. In the absence of amplification, no light signal is generated resulting in no detection of target organism. The 3M MDA 2 – Cronobacte r method allows for the rapid and specific detection of Cronobacter species in select food types and environmental surfaces after 18–24 h of pre-enrichment. After enrichment, samples are evaluated using the 3M MDA 2 – Cronobacter on the 3M Molecular Detection System (MDS). Presumptive positive results are reported in real-time and negative results are displayed after completion of the 60 min run. Prior to the collaborative study, the 3MMDA2 – Cronobacter method was validated according to AOAC Guidelines (4) in a harmonized AOAC Performance Tested Method SM (PTM) study. The assay was awarded PTM 101703 on October 24, 2017. The objective of these studies was to demonstrate that the 3M MDA 2 – Cronobacter method could detect Cronobacter in select food matrixes and environmental surfaces as claimed by the manufacturer. The following matrixes were evaluated: powdered infant formula with probiotics (10 and 300 g), powdered infant cereal without probiotics (10 and 300 g), lactose powder (10 g), and environmental surface sponges (stainless steel). The purpose of this collaborative study was to compare the reproducibility of the 3M MDA 2 – Cronobacter method with ISO 22964:2017 (5).

Received July 19, 2018. Accepted by AH August 10, 2018. This method was approved by the AOAC Expert Review Panel for Cronobacter as First Action. The Expert Review Panel invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit-for-purpose and is critical for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. Supplemental information is available online at: http://aoac. publisher.ingentaconnect.com/content/aoac/jaoac 1 Corresponding author’s e-mail: lmonteroso@mmm.com indicating that the difference between methods was not statistically significant at the 0.05 probability level. C  ronobacter, originally defined as a species, Enterobacter sakazakii , is a motile Gram-negative bacterium that causes foodborne illness (1). The opportunistic pathogenic organism has been linked with serious illness among infants, notably following the consumption of powdered infant The 3M™ Molecular Detection Assay (MDA) 2 – Cronobacter combines the use of loop-mediated isothermal amplification to rapidly amplify nucleic acid sequences while using bioluminescence to detect the amplification. Using a paired study design, the MDA 2 – Cronobacter was compared with ISO 22964:2017 for the detection of Cronobacter species in powdered infant formula containing probiotics. Technicians from 11 laboratories from the United States, Mexico, and Croatia participated. Collaborators received test portions with three levels of contamination. Statistical analysis was conducted according to the probability of detection (POD) statistical model. Results obtained for the low-inoculum level test portions produced a difference in POD values obtained from combining all valid collaborator POD data values with 95% confidence intervals of –0.01, –0.12, and 0.10,

DOI: https://doi.org/10.5740/jaoacint.18-0233

B ird et al . : J ournal of AOAC I nternational V ol . 102, N o . 1, 2019  109

Collaborative Study

Test Portion Distribution

All samples were labeled with a randomized, blind-coded three-digit number affixed to the sample container. Test portions were shipped on a Wednesday via overnight delivery according to the Category B Dangerous Goods shipment regulations set forth by the International Air Transport Association. Upon receipt, samples were held by the collaborating laboratory at ambient temperature (20–25°C) until the following Monday when analysis was initiated. In addition to each of the test portions and a separate aerobic plate count sample, collaborators received a data logger. Data loggers were programmed to monitor the temperature of the shipment to ensure it did not fall outside the range of 18–25°C. Participants were instructed to notify the study sponsor if the red alarm light was illuminated on the data logger upon receipt of the sample, which would indicate that the temperature fell outside the target temperature range (18–25°C). The information retrieved from the units were recorded on the “Sample Receipt Confirmation” form provided and faxed or emailed back to the study director. No laboratories reported temperatures outside the target range. Collaborators were instructed to follow the appropriate preparation and analysis as outlined in the study protocol. For this paired study design, each collaborator received 36 test portions (12 high, 12 low, and 12 uninoculated controls) preweighed to 10 g in sterile filter stomacher bags. Participants were instructed to add 90 mL of buffered peptone water (BPW) International Organization for Standardization (ISO) formulation (8; BPW-ISO), homogenize by stomaching or hand massaging for 2 min, and incubate at 37 ± 1°C for 18–20 h. Following enrichment, samples were assayed by the 3MMDA 2 – Cronobacter method and, regardless of presumptive result, confirmed following the ISO 22964:2017 reference method, beginning with a transfer to secondary enrichment [ Cronobacter Screening Broth (CSB)]. CSB tubes were incubated at 41.5 ± 2°C for 24 ± 2 h. After incubation, all tubes were streaked on Cronobacter Chromogenic Isolation Agar (CCI). CCI plates were incubated at 41.5 ± 2°C for 24 ± 2 h. Plates were observed for typical colonies (small to medium-sized and blue to blue-green in color), and if present, a well-isolated colony was streaked on TSA and incubated at 37 ± 2°C for 18–24 h. Isolates were confirmed positive by having a negative oxidase test and biochemical tests using API 20 E or VITEK 2 GN biochemical identification test (AOAC Official Method 2011.17 ; 9). Each collaborating laboratory reported results on the data sheets provided. The data sheets were submitted to the study director at the end of testing for statistical analysis. Data for each contamination level was analyzed using the probability of detection (POD) statistical model (10) and conducted using the AOAC Binary Data Interlaboratory Study Workbook, Version 2.3 (11). For each laboratory, the POD was calculated for the candidate presumptive results (POD CP ), the candidate confirmatory results [including false negative (FN) results; Test Portion Analysis Statistical Analysis

Study Design In this collaborative study, one matrix, powdered infant formula (milk-based with iron and docosahexaenoic acid) containing probiotics ( Lactobacillus reuteri ), was evaluated. The matrix was obtained from a local retailer and screened negative for the presence of Cronobacter by the ISO 22964:2017 reference method and by the 3MMDA2 – Cronobacter method. The matrix was artificially contaminated with a lyophilized culture of Cronobacter sakazakii , Q Laboratories isolate 17031.4 (origin: powdered infant formula), at two inoculation levels, a high inoculation level of approximately 2–5 colony-forming units (CFU)/test portion and a low inoculation level of approximately 0.2–2 CFU/test portion. A set of uninoculated control test portions (0 CFU/test portion) was also included. The 3M MDA 2 – Cronobacter and ISO 22964:2017 share a common pre-enrichment; therefore, the study was designed using paired samples. A total of 36 samples were evaluated per collaborator. Twelve replicate samples from each of the three inoculation levels were evaluated by 14 analysts from 11 locations. Collaborators were also sent a test portion for determining the total aerobic plate count (APC) using the ISO 4833-1:2013 (6) reference method on the day samples were received. A detailed collaborative study packet outlining all necessary information related to the study including media preparation, test portion preparation, and documentation of results was sent to each collaborating laboratory prior to the initiation of the study. The C. sakazakii isolate used in this evaluation was lyophilized prior to inoculation. The culture was propagated onto Trypticase Soy Agar with 5% sheep blood (TSA-SB) from a Q Laboratories frozen stock culture stored at –70°C. To prepare the culture for lyophilization, a single, well-isolated colony from TSA-SB was transferred into brain heart infusion broth and incubated at 37 ± 2°C for 18–24 h. The culture was diluted in a sterile cryoprotectant, reconstituted 10% nonfat dry milk, and freeze dried for 48–72 h. A bulk lot of the test matrix was inoculated with the culture at a high level expected to yield all positive results. The bulk lot was placed into a large stainless-steel container and mixed with sterile spatula for 30 ± 1 min. An aliquot of the high-level inoculum was further mixed in the same manner with uninoculated powdered infant formula to produce the low-level inoculum. After inoculation, the matrix was held for a minimum of 2 weeks at ambient temperature (20–25°C). The inoculated test product was packaged into separate 10 g samples in sterile Whirl-Pak ® bags and shipped to the collaborators. To determine the level of Cronobacter in the matrixes, a 5-tube most probable number (MPN) was conducted by the coordinating laboratory on the day of the initiation of analysis using the ISO 22964:2017 reference method. The MPN was determined by analyzing 5 × 20 g test portions, the reference method test portions from the collaborating laboratories 12 × 10 and 5 × 5 g test portions. The MPN and 95% confidence intervals were calculated using the Least Cost Formulations MPN Calculator, Version 1.6, provided by AOAC Research Institute (7). Preparation of Inocula and Test Portions

110  B ird et al . : J ournal of AOAC I nternational V ol . 102, N o . 1, 2019

( a )  3M Molecular Detection System (MDS100) .—Available from 3M Food Safety. ( b )  3M Molecular Detection Assay 2 – Cronobacter reagent tubes .—12 strips of eight tubes. Available from 3M Food Safety. ( c )  Lysis solution (LS) tubes .—12 strips of eight tubes. ( d )  Extra caps .—12 strips of eight caps. ( e )  Reagent control .—Eight reagent tubes. ( f )  Quick start guide. ( g )  3M Molecular Detection Speed Loader Tray. —Available from 3M Food Safety. ( h )  3M Molecular Detection Chill Block Insert. —Available from 3M Food Safety. ( i )  3M Molecular Detection Heat Block Insert.—Available from 3M Food Safety. ( j )  3M Molecular Detection Cap/Decap Tool for reagent tubes. —Available from 3M Food. ( k )  3M Molecular Detection Cap/Decap Tool for lysis tubes. —Available from 3M Food Safety. ( l )  Empty lysis tube rack .—Available from 3M Food Safety. ( m )  Empty reagent tube rack .—Available from 3M Food Safety. ( n )  BPW-ISO formulation .—Available from 3MFood Safety. ( o )  Micropipet .—Capable of 20 μL. ( p )  Multichannel (8-channel) pipet .—Capable of 20 μL. ( q )  Sterile filter pipet tips —Capable of 20 μL. ( r )  Filter Stomacher ® bags.—Seward or equivalent. ( s )  Stomacher .—Seward or equivalent. ( t )  Thermometer .—Calibrated range to include 100 ± 1°C. ( u )  Dry block heater unit .—Capable of maintaining 100 ± 1°C. ( v )  Incubators .—Capable of maintaining 37 ± 1°C. ( w )  Refrigerator .—Capable of maintaining 2–8°C for storing the 3M MDA components. ( x )  Computer .—Compatible with the 3M Molecular Detection Instrument. ( y )  3M Hydrated Sponge Stick .—Available from 3M Food Safety. C. General Instructions ( a ) Store the 3M MDA 2 – Cronobacter at 2–8°C. Do not freeze. Keep kit away from light during storage. After opening the kit, check that the foil pouch is undamaged. If the pouch is damaged, do not use. After opening, unused reagent tubes should always be stored in the resealable pouch with the desiccant inside to maintain stability of the lyophilized reagents. Store resealed pouches at 2–8°C for no longer than 60 days. Do not use 3M MDA 2 – Cronobacter past the expiration date. ( b ) Follow all instructions carefully. Failure to do so may lead to inaccurate results. ( c ) The 3M MDA 2 – Cronobacter is intended for use in a laboratory environment by professionals trained in laboratory techniques. 3M has not documented the use of this product in industries other than the food and beverage industries. For example, 3M has not documented this product for testing drinking water, pharmaceutical, cosmetics, clinical, or veterinary samples. The 3M MDA 2 – Cronobacter has not been evaluated with all possible food products, food processes, testing protocols, or with all possible strains of bacteria. ( d )As with all test methods, the source of enrichment medium can influence the results. The 3M MDA 2 – Cronobacter has only been evaluated for use with the enrichment media specified in the Instructions for Use section.

POD CC ], presumptive candidate results that confirmed positive (excluding FN results; POD C ), and the reference method (POD R ). POD values obtained from combining all valid collaborator POD data (LPOD) were determined for candidate presumptive results (LPOD CP ) candidate confirmed results (including FN results; LPOD CC ), presumptive candidate results that confirmed positive (excluding FN results; LPOD C ), and reference method results (LPOD R ). Using LPOD values, the difference in the candidate presumptive and confirmatory results from all collaborators (dLPOD CP ) and the difference in the confirmed candidate and reference methods (dLPOD C ) were calculated. A dLPOD C confidence interval not containing the point zero would indicate a statistically significant difference between the 3MMDA2 – Cronobacter method and the reference method at the 5% probability level. In addition to POD and LPOD values, the repeatability SD (s r ), the among laboratory repeatability SD (s L ), the reproducibility SD (s R ), and the P value were calculated. The s r provides the SD of data within one laboratory, the s L provides the SD among laboratories, and the s R provides the SD in data between laboratories. The P value provides information on the homogeneity of the test portions sent to the laboratories. Additionally, false positive (FP) and FN fractions were determined. FP fractions were calculated as the number of FP divided by the number of negative agreements (presumptive negative sample that confirmed negative) multiplied by 100. FN fractions were calculated by the number of FN divided by the number of positive agreements (presumptive positive sample that confirmed positive). [Applicable to detection of Cronobacter species in powdered infant formula with probiotics (10 and 300 g), powdered infant cereal without probiotics (10 and 300 g), lactose powder (10 g), and environmental surface sponges (stainless steel).] See Table 2018.01A for a summary of results of the collaborative study. See Table 2018.01B for detailed results of the collaborative study. A. Principle The 3M MDA 2 – Cronobacter method is used with the 3M MDS for the rapid and specific detection of Cronobacter species in select enriched food and food process environmental samples. The 3M MDA 2 – Cronobacter uses LAMP of unique DNA target sequences with high specificity and sensitivity combined with bioluminescence to detect the amplification. Presumptive positive results are reported in real-time, and negative results are displayed after the assay is completed. Samples are pre- enriched in BPW-ISO formulation. B. Apparatus and Reagents Items B(b–g) are available as the 3M MDA 2 – Cronobacter kit from 3M Food Safety (St. Paul, MN). AOAC Official Method 2018.01 Cronobacter species in Select Foods and Environmental Surfaces 3M ™ Molecular Detection Assay (MDA) 2 – Cronobacter Method First Action 2018

B ird et al . : J ournal of AOAC I nternational V ol . 102, N o . 1, 2019  111

Table 2018.01A. Summary of results for the detection of Cronobacter in powdered infant formula with probiotics Method a Inoculation level

3M MDA 2 – Cronobacter

Uninoculated

Low

High

Candidate presumptive positive/total No. of samples analyzed

2/168

88/168

168/168

LPOD CP

0.01 (0.01, 0.04) 0.11 (0.10, 0.15) 0.00 (0.00, 0.04) 0.11 (0.10, 0.12)

0.52 (0.45, 0.60) 0.51 (0.46, 0.52) 0.00 (0.00, 0.15) 0.51 (0.46, 0.52)

1.00 (0.98, 1.00) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

s r

s L s R

P value b

0.5158

0.7750 86/168

1.0000 168/168

Candidate confirmed positive/total No. of samples analyzed

0/168

0.00 (0.00, 0.02) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

0.51 (0.43, 0.59) 0.51 (0.46, 0.52) 0.00 (0.00, 0.11) 0.51 (0.47, 0.52)

1.00 (0.98, 1.00) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

LPOD CP

s r

s L s R

P value

1.0000

0.9494 84/168

1.0000 168/168

Candidate confirmed positive/total No. of samples analyzed Candidate presumptive positive that confirmed LPOD C

0/168

0.00 (0.00, 0.02) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

0.50 (0.42, 0.58) 0.51 (0.46, 0.52) 0.00 (0.00, 0.13) 0.51 (0.47, 0.52)

1.00 (0.98, 1.00) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

s r

s L s R

P value

1.0000

0.9462 86/168

1.0000 168/168

Positive ref. samples/total No. of samples analyzed

0/168

Ref. LPOD

0.00 (0.00, 0.02) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

0.51 (0.43, 0.59) 0.51 (0.46, 0.52) 0.00 (0.00, 0.11) 0.51 (0.47, 0.52)

1.00 (0.98, 1.00) 0.00 (0.00, 0.15) 0.00 (0.00, 0.15) 0.00 (0.00, 0.21)

s r

s L s R

P Value

1.0000

0.9494

1.0000

dLPOD (candidate vs. ref.) c

0.00 (–0.02, 0.02) 0.01 (–0.01, 0.04)

–0.01 (–0.12, 0.10) 0.01 (–0.10, 0.12)

0.00 (–0.02, 0.02) 0.00 (–0.02, 0.02)

dLPOD (candidate presumptive vs. candidate confirmed) c

a  Results include 95% confidence intervals. b P value = Homogeneity test of laboratory PODs. c  A confidence interval for dLPOD that does not contain the value 0 indicates a statistically significant difference between the two methods.

Safety Precautions

To reduce the risks associated with exposure to chemicals and biohazards, perform pathogen testing in a properly equipped laboratory under the control of trained personnel. Always follow standard laboratory safety practices, including wearing appropriate protective apparel and eye protection while handling reagents and contaminated samples. Avoid contact with the contents of the enrichment media and reagent tubes after amplification. Dispose of enriched samples according to current industry standards. After use, the enrichment medium and the 3M MDA 2 – Cronobacter tubes can potentially contain pathogenic materials. Periodically decontaminate laboratory benches and equipment (pipets, cap/decap tools, etc.) with a 1–5% (v/v in water) household bleach solution (5250–6500 ppm) or DNA removal solution. When testing is complete, follow current industry standards for the disposal of contaminated waste. Consult the Safety Data Sheet for additional information and local regulations for disposal. To reduce the risks associated with environmental contamination, follow current industry standards for disposal of contaminated waste.

The 3M Molecular Detection Instrument is intended for use with samples that have undergone heat treatment during the assay lysis step, which is designed to destroy organisms present in the sample. Samples that have not been properly heat treated during the assay lysis step may be considered a potential biohazard and should not be inserted into the 3MMDS instrument. The user should not exceed the recommended temperature setting on the heater or the recommended heating time. Use an appropriate, calibrated thermometer to verify the 3M Molecular Detection Heat Block Insert temperature (e.g., a partial immersion thermometer or digital thermocouple thermometer, not a total immersion thermometer). The thermometer must be placed in the designated location in the 3M Molecular Detection Heat Block Insert. The user should read, understand, and follow all safety information in the instructions for the 3M MDS and the 3M MDA 2 – Cronobacter . Retain the safety instructions for future reference.

112  B ird et al . : J ournal of AOAC I nternational V ol . 102, N o . 1, 2019

Table 2018.01B. Comparative results for the detection of Cronobacter in 10 g powdered infant formula with probiotics test portions by the 3M MDA 2 – Cronobacter method versus ISO 22964:2017 in a collaborative study

CP a

CC b

C c

R d

C vs. R

dLPOD (C, R)

dLPOD (CP, CC)

N e X f POD

Inoculation level Statistic

Matrix

Lab

CP N X POD CC N X POD C N X POD R

Uninoculated control

Powdered infant formula 1 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 2 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 3 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 4 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 5 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 6 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 7 12 1 0.08 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.08 8 12 1 0.08 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.08 9 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 10 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 11 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 12 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 13 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 14 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 Estimate All 168 2 0.01 168 0 0.00 168 0 0.00 168 0 0.00 0.00 0.01 LCL g 0.01 0.00 0.00 0.00 –0.02 –0.01 UCL h 0.04 0.02 0.02 0.02 0.02 0.04 s r 0.11 0.00 0.00 0.00 LCL 0.10 0.00 0.00 0.00 UCL 0.15 0.15 0.15 0.15 s L 0.00 0.00 0.00 0.00 LCL 0.00 0.00 0.00 0.00 UCL 0.04 0.15 0.15 0.15 s R 0.11 0.00 0.00 0.00 UCL 0.10 0.00 0.00 0.00 LCL 0.12 0.21 0.21 0.21 P value 0.5158 1.0000 1.0000 1.0000 Powdered infant formula 1 12 6 0.50 12 6 0.50 12 6 0.50 12 6 0.50 0.00 0.00 2 12 7 0.58 12 7 0.58 12 7 0.58 12 7 0.58 0.00 0.00 3 12 5 0.42 12 6 0.50 12 5 0.42 12 6 0.00 –0.08 –0.08 4 12 5 0.42 12 5 0.42 12 5 0.42 12 5 0.42 0.00 0.00 5 12 4 0.33 12 4 0.33 12 4 0.33 12 4 0.33 0.00 0.00 6 12 5 0.42 12 5 0.42 12 5 0.42 12 5 0.42 0.00 0.00 7 12 8 0.66 12 6 0.50 12 6 0.50 12 6 0.50 0.00 0.16 8 12 6 0.50 12 6 0.50 12 6 0.50 12 6 0.50 0.00 0.00 9 12 6 0.50 12 6 0.50 12 6 0.50 12 6 0.50 0.00 0.00 10 12 5 0.42 12 5 0.42 12 5 0.42 12 5 0.42 0.00 0.00 MPN/ test portion (0.70, 0.57, 0.87) 11 12 8 0.66 12 8 0.66 12 8 0.66 12 8 0.66 0.00 0.00 12 12 7 0.58 12 7 0.58 12 6 0.50 12 7 0.58 –0.08 0.08 13 12 9 0.75 12 8 0.66 12 8 0.66 12 8 0.66 0.00 0.08 14 12 7 0.58 12 7 0.58 12 7 0.58 12 7 0.58 0.00 0.00

Low

Estimate

All 168 88 0.52 168 86 0.51 168 84 0.50 168 86 0.51 –0.01 0.01

LCL UCL

0.45 0.60 0.51 0.46 0.52

0.43 0.59 0.51 0.46 0.52

0.42 0.58 0.51 0.46 0.52

0.43 –0.12 –0.10 0.59 0.10 0.12

0.51 0.46 0.52

s r

LCL UCL

B ird et al . : J ournal of AOAC I nternational V ol . 102, N o . 1, 2019  113

Table 2018.01B. ( continued )

CP a

CC b

C c

R d

C vs. R

dLPOD (C, R)

dLPOD (CP, CC)

N e X f POD

Inoculation level Statistic

Matrix

Lab

CP N X POD CC N X POD C N X POD R

0.00 0.00 0.15 0.51 0.46 0.52

0.00 0.00 0.12 0.51 0.47 0.52

0.00 0.00 0.13 0.51 0.47 0.52

0.00 0.00 0.12 0.51 0.47 0.52

s L

LCL UCL

s R

UCL LCL

P value

0.7750

0.9494

0.9462

0.9494

High

Powdered infant formula 1 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 2 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 3 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 4 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 5 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 6 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 7 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 8 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 9 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 10 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 MPN/ test portion (2.71, 1.86, 3.97) 11 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 12 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 13 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 14 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00

Estimate

168 168 1.00 168 168 1.00 168 168 1.00 168 168 1.00 0.00 0.00

LCL UCL

0.98 1.00 0.00 0.00 0.15 0.00 0.00 0.15 0.00 0.00 0.21

0.98 1.00 0.00 0.00 0.15 0.00 0.00 0.15 0.00 0.00 0.21

0.98 1.00 0.00 0.00 0.15 0.00 0.00 0.15 0.00 0.00 0.21

0.98 –0.02 –0.02 1.00 0.02 0.02

0.00 0.00 0.15 0.00 0.00 0.15 0.00 0.00 0.21

s r

LCL UCL

s L

All

LCL UCL

s R

UCL LCL

P value

1.000

1.000

1.000

1.000

a  CP = Candidate presumptive. b  CC = Candidate confirmed.

c  C = Candidate result. d  R = Reference method. e  N = Number of samples tested. f  X = Number of test samples with a positive result (presumptive or confirmed).

g  LCL = Lower confidence limit. h  UCL = Upper confidence limit.

114  B ird et al . : J ournal of AOAC I nternational V ol . 102, N o . 1, 2019

G. Preparation of the 3M Molecular Detection Heat Block Insert Place the 3M Molecular Detection Heat Block Insert in a dry double block heater unit. Turn on the dry block heater unit and set the temperature to allow the 3M Molecular Detection Heat Block Insert to reach and maintain a temperature of 100 ± 1°C. Note: Depending on the heater unit, allow approximately 30 min for the 3M Molecular Detection Heat Block Insert to reach temperature. Using an appropriate, calibrated thermometer (e.g., a partial immersion thermometer, digital thermocouple thermometer, not a total immersion thermometer) placed in the designated location, verify that the 3M Molecular Detection Heat Block Insert is at 100 ± 1°C. H. Preparation of the 3M Molecular Detection Instrument ( a ) Launch the 3MMolecular Detection Software and log in. Contact your 3M Food Safety representative to ensure you have the most updated version of the software. ( b ) Turn on the 3M Molecular Detection Instrument. ( c ) Create or edit a run with data for each sample. Refer to the 3M MDS User Manual for details. Note: The 3M Molecular Detection Instrument must reach and maintain temperature of 60°C before inserting the 3M Molecular Detection Speed Loader Tray with reaction tubes. This heating step takes approximately 20 min and is indicated by an ORANGE light on the instrument’s status bar. When the instrument is ready to start a run, the status bar will turn GREEN. H. Lysis ( a )  Allow the LS tubes to warm up by setting the rack at room temperature (20–25°C) overnight (16–18 h) .—Alternatives to equilibrate the LS tubes to room temperature are to set the LS tubes on the laboratory bench for at least 2 h, incubate the LS tubes in a 37 ± 1°C incubator for 1 h, or place them in a dry double block heater for 30 s at 100 ± 1°C. ( b ) Invert the capped tubes to mix. Proceed to the next step within 4 h after inverting. ( c ) Remove the enrichment broth from the incubator. ( d )  One LS tube is required for each sample and the negative control (NC; sterile enrichment medium) sample.—(1) LS tube strips can be cut to desired LS tube number. —Select the number of individual LS tubes or 8-tube strips needed. Place the LS tubes in an empty rack. ( 2 ) To avoid cross-contamination, decap one LS tube strip at a time and use a new pipet tip for each transfer step. ( 3 ) Transfer enriched sample to LS tubes. Transfer each enriched sample into individual LS tube first. Transfer the NC last. ( 4 ) Use the 3M Molecular Detection Cap/Decap Tool-Lysis to decap one LS tube strip one strip at a time. ( 5 ) Discard the LS tube cap. If lysate will be retained for retest, place the caps into a clean container for reapplication after lysis. ( 6 ) Agitate the enrichment bag before collecting the sample from the filtered side when working with viscous samples. ( 7 ) Transfer 20 μLsample into a LS tube ( see Figure 2018.01A ). ( e ) Repeat steps 1–4 as needed for the number of samples to be tested. When all samples have been transferred, then transfer 20 μL NC into a LS tube. Do not recap tubes.

D. Sample Enrichment

Food Matrixes ( a ) Allow the BPW-ISO to equilibrate to ambient laboratory temperature (20–25°C) for 10 g test portions or environmental samples or to 37°C for 300 g test portions. ( b )  Enrich samples following a 1:9 enrichment ratio .— ( 1 ) For example, to 10 g test portions, a 90 mL volume of BPW-ISO is added. ( 2 ) For 300 g powdered infant formula and powdered infant cereal with probiotics, 10 mg/L Vancomycin is required to be supplemented into 2700 mL BPW-ISO. ( c ) Homogenize thoroughly by blending, stomaching, vortex mixing, or hand mixing for 2 ± 0.2 min, or until all lumps are completely dissolved and the enrichment suspension is homogeneous. ( d )  Incubation .—( 1 ) Incubate powdered infant formula and powdered infant cereal (10 g) for 18–20 h at 37 ±1°C. ( 2 ) Incubate powdered infant formula nonprobiotic (300 g) for 18–24 h at 37 ± 1°C. ( 3 ) Incubate powdered infant formula and powdered infant cereal with probiotics (300 g) for 22–24 h at 37 ± 1°C. ( 4 ) Incubate lactose (10 g) for 18–24 h at 37 ±1°C. ( a ) Sample collection devices should be a sponge-hydrated with Dey-Engley Neutralizing Broth. It is recommended to sanitize the area after sampling. ( b ) The recommended size of the sampling area to verify the presence or absence of the pathogen on the surface is at least 100 cm 2 (10 × 10 cm or 4 × 4 in.). When sampling with a sponge, cover the entire area going in two directions (left to right then up and down) or collect environmental samples following current sampling protocol or ISO 18593:2004 (12) guidelines. ( c ) Allow the BPW-ISO to equilibrate to ambient laboratory temperature (20–25°C). ( d ) Enrich samples by adding a 90 mL volume BPW-ISO to a sampling sponge. ( e ) Homogenize thoroughly by stomaching or hand mixing for 2 ± 0.2 min. Incubate at 37 ± 1°C for 18–24 h. E. Preparation of the 3M Molecular Detection Speed Loader Tray ( a ) Wet a cloth or paper towel with a 1–5% (v/v in water) household bleach (5250–6500 ppm) solution and wipe the 3M Molecular Detection Speed Loader Tray. ( b ) Rinse the 3M Molecular Detection Speed Loader Tray with water. ( c ) Use a disposable towel to wipe the 3M Molecular Detection Speed Loader Tray dry. ( d ) Ensure the 3M Molecular Detection Speed Loader Tray is dry before use. F. Preparation of the 3M Molecular Detection Chill Block Insert Place the 3M Molecular Detection Chill Block Insert directly on the laboratory bench; the 3M Molecular Detection Chill Block Tray is not used. Use the block at ambient laboratory temperature (20–25°C). Environmental Samples