4. AOACRIMicroMethods-2018Awards

201 9 AOAC OFFICIAL METHODS BOARD AWARDS 

201 6  ‐ 201 8 RESEARCH INSTITUTE MICROBIOLOGY METHODS TO BE  REVIEWED FOR   201 9 METHOD OF THE YEAR  OFFICIAL METHODS OF ANALYSIS OF AOAC INTERNATIONAL 

METHOD OF THE YEAR  OMB may select more than one method in this category each year.  

Selection Criteria  The minimum criteria for selection are: 

a. The method must have been approved for first or final action within the last three years. b. Generally, some unique or particularly noteworthy aspect of the method is highlighted as making it worthy of the award, such as innovative technology or application, breadth of applicability, critical need, difficult analysis, and/or range of collaborators. c. The method demonstrates significant merit in scope or is an innovative approach to an analytical problem. Selection Process:  a. AOAC staff lists all eligible methods for consideration and forwards that list with supporting documentation (e.g. ERP chair recommendation(s)) to the Chair of the Official Methods Board (OMB). b. The Chair forwards the list along with any supporting information to the members of the OMB. c. The OMB selects the Method of the Year. The winner is selected by 2/3 vote. If necessary, the OMB chair may cast tie‐breaking vote. Award  An appropriate letter of appreciation and thanks will be sent to the author(s) of the winning  method. The corresponding author will be announced at the appropriate session of the AOAC  INTERNATIONAL annual meeting, with presentation of an award. All authors will be acknowledged  at the annual meeting, will receive an award and a letter of appreciation. The name of the  winner(s), with supporting story, will be carried in the announcement in the  ILM .

TABLE OF CONTENTS FOR METHODS  MICROBIOLOGY SOLE‐SOURCE OR PROPRIETARY METHODS REVIEWED IN 2016 – 2018

AOAC 2016.01  Salmonella  spp. in Select Foods and Environmental Surfaces 

AOAC 2016.07  Listeria  in Select Foods and Environmental Surfaces 

22 

AOAC 2016.08  Listeria monocytogenes  in Variety of Foods and Select Environmental 

39 

Surfaces 

AOAC 2015.13  Enumeration of Aerobic Bacteria in Food 

54 

AOAC 2014.01  Salmonella  in Selected Foods 

67 

AOAC 2014.05  Enumeration of Yeast and Mold in Food 

80 

AOAC 2014.06  Listeria  species in Selected Foods and Environmental Surfaces 

102 

AOAC 2014.07  Listeria monocytogenes  in Selected Foods and Environmental Surfaces 

117 

AOAC 2013.01  Salmonella  in a Variety of Foods and Environmental Surfaces 

130 

AOAC 2013.02  Salmonella  species in a Variety of Foods 

144 

AOAC 2013.09  Salmonella  in Selected Foods 

152 

AOAC 2013.10  Listeria  in a Variety of Foods and Environmental Surfaces  (2014 Multi‐ Laboratory Study of the Year – Microbiology – join with 2013.11) AOAC 2013.11  Listeria monocytogenes  in Variety of Foods  (2014 Multi‐Laboratory Study of the Year – Microbiology – join with 2013.10) AOAC 2013.14  Identification of  Salmonella  spp. From Colony Picks  (2014 Award in Recognition of Technical and Scientific Excellence for co‐authors) AOAC 2012.02  Gram Positive Microbial Identification  ( ERP Chair Nomination for 2017

163 

174 

185 

193 

Method of the Year)

AOAC 2017.01  Escherichia coli O157:H7 in Selected Foods 

201  236 

AOAC 2017.05 

Escherichia coli O157:H7 and Escherichia coli non‐O157 Shiga Toxin‐  Producing Escherichia coli (STEC) in Select Foods 

AOAC 2017.06  Salmonella in Select Foods 

270  308

AOAC 2017.09 

Confirmation and Identification of Salmonella species, Cronobacter  species, and Other Gram‐Negative Organisms  (2018 Method of the Year)

AOAC 2017.10 

316 

Confirmation and Identification of Listeria monocytogenes, Listeria  species, and Other Gram‐Positive Organisms  (2018 Method of the Year)

AOAC 2018.01  Cronobacter species in Select Foods and Environmental Surfaces  322  AOAC 2018.02  Enumeration of Yeast and Mold in Select Foods MC‐Media Pad™ Yeast and  Mold Device  336  AOAC 2018.05  Enumeration of Enterobacteriaceae in Select Foods and Environmental  Surfaces  342  AOAC 2018.13  Enumeration of Escherichia coli and Coliform in a Broad Range of Foods  and Select Environmental Surfaces  348

980 B ird et al .: J ournal of aoaC i nternational V ol . 99, n o . 4, 2016 FOOD BIOLOGICAL CONTAMINANTS

4

Evaluation of the 3M™Molecular Detection Assay (MDA) 2 – Salmonella for the Detection of Salmonella spp. in Select Foods and Environmental Surfaces: Collaborative Study, First Action 2016.01 P atrick B ird , J onathan F lannery , e rin c rowley , J ames r. a gin , and d avid g oins Q Laboratories, Inc., 1400 Harrison Ave, Cincinnati, OH 45214 l isa m onteroso 3M Food Safety Department, 3M Center, Bldg 260-6B-01, St. Paul, MN 55144 Collaborators: C. Barnes; B. Bastin; J. Blumfield; T. Bonilla; R. Brooks; E. Budge; A. Calle; D. Campos; J. Casimir; N. Cuthbert; A. Deshields; Z. Geurin; C. Gies; A. Hankins; L. Hardrath; B. Kupski; M. Mendres; J. Miller; K. Naylor; J. Pickett; A. Repeck; J. Reynolds; B. Schindler; J. Schoeni; M. Tillottson; L. Thompson; H. Wright; C. Zook

The 3M™ Molecular Detection Assay (MDA) 2 – Salmonella uses real-time isothermal technology for the rapid and accurate detection of Salmonella spp. from enriched select food, feed, and food-process environmental samples. The 3M MDA 2 – Salmonella was evaluated in a multilaboratory collaborative study using an unpaired study design. The 3M MDA 2 – Salmonella was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 5 reference method for the detection of Salmonella in creamy peanut butter, and to the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapter 4.08 reference method “Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and Catfish Products and Carcass and Environmental Samples” for the detection of Salmonella in raw ground beef (73% lean). Technicians from 16 laboratories located within the continental United States participated. Each matrix was evaluated at three levels of contamination: an uninoculated control level (0 CFU/test portion), a low inoculum level (0.2–2 CFU/test portion), and a high inoculum level (2–5 CFU/test portion). Statistical analysis was conducted according to the probability of detection (POD) statistical model. Results obtained for the low inoculum level test portions produced difference in collaborator POD Submitted for publication March 25, 2016. This method was approved by the Expert Review Panel for Microbiology Methods for Food and Environmental Surfaces as First Action. The Expert Review Panel for Microbiology Methods for Food and Environmental Surfaces 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 are critical for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. Corresponding author’s e-mail: pbird@qlaboratories.com DOI: 10.5740/jaoacint.16-0085

values of 0.03 (95% confidence interval, −0.10 to 0.16) for raw ground beef and 0.06 (95% confidence interval, −0.06 to 0.18) for creamy peanut butter, indicating no statistically significant difference between the candidate and reference methods. S almonella is a nonspore-forming, rod-shaped, Gram-negative bacterium that can cause disease in humans (1). Most of the Salmonella serovars cause gastrointestinal illness, with annual estimates of over 1 million illnesses and 450 deaths in the United States (1). A few serovars, Salmonella Typhi and Salmonella Paratyphi A, B, and C, can cause typhoidal illness also known as enteric fever (2). In the past year, Salmonella has been identified as the source of over 20 food-related outbreaks in the United States (3, 4). The 3M™ Molecular Detection Assay (MDA) 2 – Salmonella method uses a combination of bioluminescence and isothermal amplification of nucleic acid sequences to rapidly detect Salmonella in select food matrixes and from environmental surfaces. The isothermal amplification is a molecular reaction conducted at a constant temperature, eliminating the need for temperature cycling and decreasing the time to results. The 3M MDA 2 – Salmonella method allows for the rapid and specific detection of Salmonella spp. in select matrixes after as little as 10 to 18 h of preenrichment using an International Organization for Standardization (ISO) formulation of buffered peptone water (BPW; 5). After enrichment, samples are evaluated using the 3MMDA2 – Salmonella on the 3MMolecular Detection System (MDS). Presumptive positive results are reported in real time, whereas negative results are displayed after completion of the assay in approximately 60 min. Before the collaborative study, the 3M MDA 2 – Salmonella method was validated according to AOAC INTERNATIONAL guidelines (6) in a harmonized AOAC Performance Tested Method SM (PTM) study. The objective of the PTM study was to demonstrate that the 3M MDA 2 – Salmonella method could detect Salmonella in select food matrixes and environmental surfaces as claimed by the manufacturer. For the 3M MDA 2 – Salmonella PTM evaluation, 18 matrixes were evaluated: raw ground beef (73% lean; 25 and 325 g), raw ground chicken (25 and 325 g), chicken carcass rinse, chicken carcass sponge,

03/10/2019

B ird et al .: J ournal of aoaC i nternational V ol . 99, n o . 4, 2016 981

5

instant nonfat dry milk (25 g), black pepper (25 g), cocoa powder (25 g), pasteurized liquid whole egg (100 g), raw head on shrimp (25 g), raw bagged spinach (25 g), spent sprout irrigation water (375 mL), cooked breaded chicken (325 g), creamy peanut butter (25 and 375 g), dry dog food (25 and 375 g), pasteurized American cheese (25 g), sealed concrete environmental surface [4 × 4 in. area with 3M hydrated sponge stick with Dey/Engeley (D/E)], stainless steel environmental surface (1 × 1 in. area with 3M Enviroswab), and sealed ceramic tile environmental surface (4 × 4 in. area with 3M hydrated sponge stick with D/E). Additional PTM parameters (inclusivity, exclusivity, ruggedness, stability, and lot-to-lot variability) tested in the PTM studies satisfied the performance requirements for PTM approval. The method was awarded PTM certification number 091501 on September 21, 2015. The purpose of this collaborative study was to compare the reproducibility of the 3M MDA 2 – Salmonella method to the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) Chapter 4.08 “Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg, and Catfish Products and Carcass and Environmental Sponges” reference method (USDA/FSIS MLG; 7) for raw ground beef (73% lean) and to the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 “ Salmonella ” reference method (FDA BAM; 8) for creamy peanut butter. In this collaborative study, two matrixes, raw ground beef (73% lean) and creamy peanut butter (50% fat, 22% sugar content), were evaluated. The matrixes were obtained from a local retailer and screened for the presence of Salmonella by the appropriate reference method before analysis. The raw ground beef was artificially contaminated with nonheat-stressed cells of Salmonella Agona, American Type Culture Collection (ATCC) 51957, and the creamy peanut butter was artificially contaminated with heat-stressed cells of Salmonella Muenchen, ATCC BAA- 1594, at two inoculation levels: a high inoculation level of approximately 2–5 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. Twelve replicate samples from each of the three inoculation levels were analyzed by each method. Two sets of samples (72 total) were sent to each laboratory for analysis by 3M MDA 2 – Salmonella and by either the USDA/FSIS MLG (raw ground beef) or FDA BAM (creamy peanut butter) reference method due to the different sample enrichment procedures for each method. In addition, collaborators were sent a 60 g test portion and instructed to conduct a total aerobic plate count (APC) using 3M Petrifilm™Aerobic Count Plate (AOAC Official Method SM 990.12 ; 9) on the day samples were received for the purpose of determining the total aerobic microbial load. 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 before the initiation of the study. A conference call was then conducted to discuss the details of the collaborative study packet and answer any questions from the participating laboratories. Collaborative Study Study Design

Preparation of Inocula and Test Portions

The Salmonella cultures used in this evaluation were propagated onto tryptic soy agar (TSA) with 5% sheep blood agar from a Q Laboratories, Inc. (Cincinnati, OH) frozen stock culture stored at −70°C. Each organism was incubated for 24 ± 2 h at 35 ± 1°C. Isolated colonies were picked to 10 mL brain heart infusion broth and incubated for 18 ± 0.5 h at 35 ± 1°C. Before inoculation, the culture suspension for the creamy peanut butter was heat-stressed at 55 ± 1°C in a water bath for 15 ± 0.5 min to obtain an injury of 50–80% [as determined by plating onto selective xylose lysine deoxycholate (XLD) agar and nonselective TSA]. The degree of injury was estimated as is the number of colonies onnonselective agar.Appropriate dilutions of each culture were prepared in Butterfield’s phosphate diluent based on previously established growth curves for both low and high inoculation levels. Bulk portions of each matrix were inoculated with the diluted liquid inoculum and mixed thoroughly to ensure an even distribution of microorganisms. The inoculated creamy peanut butter was packaged into separate 30 g test portions in sterile Whirl-Pak ® bags and shipped to the collaborators. For the analysis of the raw ground beef, 25 g inoculated test product was mixed with 300 g uninoculated test product to prepare 325 g test portions, which were packaged in sterile Whirl-Pak bags and shipped to the collaborators. To determine the level of Salmonella in the matrixes, a most probable number (MPN) assay was conducted by the coordinating laboratory on the day of the initiation of analysis using the USDA/FSIS MLG reference method for raw ground beef and the FDA BAM reference method for the creamy peanut butter. For raw ground beef, the MPN was determined by analyzing 5 × 650 g test portions, the reference method test portions from the collaborating laboratories, and 5 × 160 g test portions by the USDA/FSIS MLG reference method. For the creamy peanut butter, the MPN of the high and low inoculated levels was determined by analyzing 5 × 50 g test portions, the reference method test portions from the collaborating laboratories, and 5 × 10 g test portions by the FDA BAM reference method. The MPN and 95% confidence intervals were calculated using the Least Cost Formulations, Ltd MPN Calculator, Version 1.6 (www.lcftld.com/customer/ LCFMPNCalculator.exe), provided by the AOAC Research Institute (10). Confirmation of the samples was conducted according to the USDA/FSIS MLG or the FDA BAM reference method, depending on the matrix. All samples were labeled with a randomized, blind-coded three-digit number affixed to the sample container. Test portions were shipped on a Thursday via overnight delivery according to the Category B Dangerous Goods shipment regulations set forth by the International Air Transportations Association. Upon receipt, raw ground beef samples were held by the collaborating laboratory at refrigeration temperature (2–8°C) until the following Monday when analysis was initiated after a total equilibration −   1   × n n 100 select nonselect where n select is the number of colonies on selective agar, and n nonselect Test Portion Distribution

03/10/2019

982 B ird et al .: J ournal of aoaC i nternational V ol . 99, n o . 4, 2016 time of 96 h. All raw ground beef samples were packed with cold packs to target a temperature of <7°C during shipment. Creamy peanut butter test samples were inoculated 10 days prior to the shipment. Samples were shipped on a Thursday via overnight delivery and held at ambient temperature (20–25°C) until the following Monday when analysis was initiated after a total of 2 weeks of equilibration of the inoculum.

6

5% probability level. In addition to POD, repeatability SD (s r ), among-laboratory repeatability SD (s L ), reproducibility SD (s R ), and the homogeneity test of laboratory PODs ( P T ) value were calculated. The s r provides the variance of data within one laboratory, the s L provides the difference in SD between laboratories, and the s R provides the variance in data between different laboratories. The P T indicates if adequate sample homogeneity has occurred between laboratories (14).

In addition to each of the test portions and a separate APC sample, collaborators received a test portion for each matrix labeled as “temperature control.” Participants were instructed to obtain the temperature of this portion upon receipt of the package, document the results on the Sample Receipt Confirmation form provided, and fax or e-mail it back to the study director. The shipment and hold times of the inoculated test material had been verified as a QC measure before study initiation. Collaborators were instructed to follow the appropriate preparation and analysis protocol for eachmatrix for the 3MMDA2 – Salmonella method and the referencemethods. For bothmatrixes, each collaborator received 72 test portions (12 high, 12 low, and 12 uninoculated controls for each method to be performed). For the analysis of the raw ground beef test portions by the 3M MDA 2 – Salmonella method, a 325 g portion was enriched with 975 mL prewarmed (41.5 ± 1°C) ISO BPW, homogenized for 2 min, and incubated for 10 h at 41.5 ± 1°C. For the creamy peanut butter test portions analyzed by the 3MMDA 2 – Salmonella method, a 25 g portion was enriched with 225 mL ISO BPW, homogenized for 2 min, and incubated for 18 h at 37 ± 1°C. After enrichment, samples were assayed by the 3M MDA 2 – Salmonella method and, regardless of presumptive result, confirmed using the appropriate reference method. Both matrixes evaluated by the 3M MDA 2 – Salmonella method were compared to samples analyzed using either the USDA/ FSIS MLG or FDABAM reference method in an unpaired study design. All positive test portions were biochemically confirmed by the API 20E biochemical test (AOAC Official Method 978.24 ; 11) or by the VITEK 2 Gram-negative biochemical identification test (AOAC Official Method 2011.17 ; 12). Polyvalent Salmonella serological testing was also performed. Each collaborating laboratory recorded the results for the reference method and the 3M MDA 2 – Salmonella method on the data sheets provided. The data sheets were submitted to the study director at the end of each week of testing for statistical analysis. Data for each test portion size was analyzed using the probability of detection (POD) statistical model (13). POD was calculated as the number of positive outcomes divided by the total number of trials. The POD was calculated for the candidate presumptive results (POD CP ), the candidate confirmatory results (including false-negative results; POD CC ), the difference in collaborator POD (dLPOD) in the candidate presumptive and confirmatory results (dLPOD CP ), presumptive candidate results that confirmed positive (excluding false-negative results; POD C ), the reference method (POD R ), and the difference in the confirmed candidate and reference methods (dLPOD C ). A dLPOD C confidence interval not containing the value 0 would indicate a statistically significant difference between the 3M MDA 2 – Salmonella and the reference methods at the Test Portion Analysis Statistical Analysis

AOAC Official Method 2016.01 Salmonella spp. in Select Foods and Environmental Surfaces 3M™ Molecular Detection Assay (MDA) 2 – Salmonella Method First Action 2016

(Applicable to detection of Salmonella spp. in raw ground beef (73% lean), raw ground chicken, chicken carcass rinse, chicken carcass sponge, pasteurized liquid whole egg, cooked breaded chicken, instant nonfat dry milk, black pepper, cocoa powder, raw whole shrimp, raw bagged spinach, creamy peanut butter, dry dog food, pasteurized processed American cheese, spent sprout irrigation water, and sealed concrete, stainless steel, and sealed ceramic tile environmental surfaces.) Caution : The 3M MDA 2 – Salmonella 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 3MMDA 2 – Salmonella has not been evaluated with all possible food products, food processes, testing protocols, or with all possible strains of bacteria.

As with all test methods, the source of enrichment medium can influence the results. The 3MMDA 2 – Salmonella has only been evaluated for use with the enrichment media specified in the manufacturers instructions for use. The 3M MDS 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 read, understand, and follow all safety information in the instructions for the 3MMDS and the 3MMDA 2 – Salmonella . Retain the safety instructions for future reference. Periodically decontaminate laboratory benches and equipment (pipets, cap/decap tools, etc.) with a 1–5% (v/v in water) household bleach solution or DNAremoval solution. When testing is complete, follow current industry standards for the disposal of contaminated waste. Consult the Material Safety Data Sheet for additional information and local regulations for disposal. To reduce the risks associated with exposure to chemicals and biohazards, ( 1 ) perform pathogen testing in a properly equipped laboratory under the control of trained personnel; ( 2 ) always follow standard laboratory safety practices, including wearing appropriate protective apparel and eye protection while handling reagents and contaminated samples; ( 3 ) avoid contact with the contents of the enrichment media and reagent

03/10/2019

B ird et al .: J ournal of aoaC i nternational V ol . 99, n o . 4, 2016 983

7

tubes after amplification; and ( 4 ) dispose of enriched samples according to current industry standards. To reduce the risks associated with environmental contamination, follow current industry standards for disposal of contaminated waste. See Tables 2016.01A and 2016.01B for a summary of results of the interlaboratory study. See Tables 2016.01C and 2016.01D for detailed results of the interlaboratory study. The 3M MDA 2 – Salmonella method is used with the 3M MDS for the rapid and specific detection of Salmonella in enriched food, feed, and food-process environmental samples. The 3M MDA 2 – Salmonella uses loop-mediated isothermal amplification of unique DNA target sequences with high specificity and sensitivity, combined with bioluminescence to detect the amplification. Presumptive positive results are A. Principle

reported in real time, whereas negative results are displayed after the assay is completed. Samples are preenriched in ISO BPW.

B. Apparatus and Reagents

Items b – g are available as the 3M MDA 2 – Salmonella kit from 3M Food Safety (St. Paul, MN). (a) 3M MDS .—MDS100 (3M Food Safety). (b) 3M MDA 2 – Salmonella reagent tubes .—Twelve strips of eight tubes. (c) Lysis solution (LS) tubes .—Twelve strips of eight tubes. (d) Extra caps .—Twelve strips of eight caps. (e) Reagent control .—Eight reagent tubes. (f) Quick Start Guide . (g) 3M Molecular Detection speed loader tray . (h) 3M Molecular Detection chill block insert .—3M Food Safety.

Table 2016.01A. Summary of results for the detection of Salmonella in raw ground beef (325 g) a

Inoculation level

3M MDA 2 – Salmonella results

Uninoculated

Low

High

Candidate presumptive positive/total No. of samples analyzed

4/156

83/156

155/156

0.03 (0.01–0.06) 0.15 (0.14–0.17) 0.04 (0.00–0.09) 0.16 (0.14–0.19)

0.53 (0.44–0.62) 0.49 (0.44–0.52) 0.09 (0.00–0.24) 0.50 (0.45–0.52)

0.99 (0.96–1.00) 0.08 (0.07–0.15) 0.00 (0.00–0.03) 0.08 (0.07–0.09)

POD CP

s r

s L s R P T

0.0315

0.1725 83/156

0.4395 155/156

Candidate confirmed positive/total No. of samples analyzed

3/156

0.02 (0.01–0.06) 0.14 (0.12–0.15) 0.03 (0.00–0.07) 0.14 (0.12–0.15)

0.53 (0.43–0.63) 0.49 (0.44–0.52) 0.12 (0.00–0.27) 0.50 (0.45–0.52)

0.99 (0.96–1.00) 0.08 (0.07–0.15) 0.00 (0.00–0.03) 0.08 (0.07–0.09)

POD CC

s r

s L s R P T

0.0877

0.0715 82/156

0.4395 155/156

Candidate confirmed positive/total No. of samples analyzed

2/156

0.01 (0.01–0.05) 0.11 (0.10–0.15) 0.03 (0.01–0.07) 0.11 (0.10–0.13)

0.53 (0.43–0.62) 0.49 (0.44–0.52) 0.10 (0.00–0.25) 0.50 (0.45–0.52)

0.99 (0.96–1.00) 0.08 (0.07–0.15) 0.00 (0.00–0.03) 0.08 (0.07–0.09)

POD C

s r

s L s R P T

0.0184

0.1272 77/156

0.4395 156/156

Positive reference samples/total No. of samples analyzed

2/156

0.01 (0.00–0.05) 0.11 (0.10–0.15) 0.00 (0.00–0.04) 0.11 (0.10–0.13)

0.49 (0.41–0.58) 0.49 (0.45–0.52) 0.06 (0.00–0.22) 0.50 (0.45–0.52)

1.00 (0.98–1.00) 0.00 (0.00–0.15) 0.00 (0.00–0.15) 0.00 (0.00–0.21)

POD R

s r

s L s R P T

0.5167

0.2813

1.0000

(candidate versus

0.00 (−0.03 to 0.03)

0.03 (−0.10 to 0.16)

−0.01 (−0.04 to 0.02)

dLPOD C

reference) b

dLPOD CP (candidate presumptive versus candidate confirmed) b

0.01 (−0.03 to 0.05)

0.00 (−0.14 to 0.14)

0.00 (−0.03 to 0.03)

a Results include 95% confidence intervals. b A confidence interval for dLPOD that does not contain the value 0 indicates a statistical significant difference between the two methods .

03/10/2019

984 B ird et al .: J ournal of aoaC i nternational V ol . 99, n o . 4, 2016

8

Table 2016.01B. Summary of results for the detection of Salmonella in creamy peanut butter (25 g)

Inoculation level

3M MDA 2 – Salmonella results a Candidate presumptive positive/total No. of samples analyzed

Uninoculated

Low

High

3/144

83/144

144/144

0.02 (0.01–0.06) 0.14 (0.12–0.16) 0.03 (0.00–0.08) 0.14 (0.13–0.17)

0.58 (0.49–0.66) 0.50 (0.45–0.52) 0.00 (0.00–0.16) 0.50 (0.45–0.52)

1.00 (0.97–1.00) 0.00 (0.00–0.16) 0.00 (0.00–0.16) 0.00 (0.00–0.22)

POD CP

s r

s L s R P T

0.0976

0.7877 82/144

1.0000 144/144

Candidate confirmed positive/total No. of samples analyzed

2/144

0.01 (0.00–0.05) 0.11 (0.10–0.16) 0.04 (0.01–0.07) 0.12 (0.11–0.14)

0.57 (0.49–0.65) 0.51 (0.46–0.52) 0.00 (0.00–0.12) 0.51 (0.46–0.52)

1.00 (0.97–1.00) 0.00 (0.00–0.16) 0.00 (0.00–0.16) 0.00 (0.00–0.22)

POD CC

s r

s L s R P T

0.0221

0.9709 81/144

1.0000 144/144

Candidate confirmed positive/total No. of samples analyzed

1/144

0.01 (0.00–0.04) 0.08 (0.07–0.16) 0.00 (0.00–0.03) 0.08 (0.07–0.10)

0.56 (0.48–0.65) 0.51 (0.45–0.52) 0.00 (0.00–0.13) 0.51 (0.46–0.52)

1.00 (0.97–1.00) 0.00 (0.00–0.16) 0.00 (0.00–0.16) 0.00 (0.00–0.22)

POD C

s r

s L s R P T

0.4368

0.9393 73/144

1.0000 144/144

Positive reference samples/total No. of samples analyzed

0/144

0.00 (0.00–0.03) 0.00 (0.00–0.16) 0.00 (0.00–0.16) 0.00 (0.00–0.22)

0.51 (0.42–0.59) 0.51 (0.46–0.52) 0.00 (0.00–0.14) 0.51 (0.46–0.52)

1.00 (0.97–1.00) 0.00 (0.00–0.16) 0.00 (0.00–0.16) 0.00 (0.00–0.22)

POD R

s r

s L s R P T

1.0000

0.8963

1.0000

(candidate versus

0.01 (−0.02 to 0.04)

0.06 (−0.06 to 0.17)

0.00 (−0.03 to 0.03)

dLPOD C

reference) b

dLPOD CP (candidate presumptive versus candidate confirmed) b

0.01 (−0.03 to 0.05)

0.01 (−0.11 to 0.13)

0.00 (−0.03 to 0.03)

a Results include 95% confidence intervals. b A confidence interval for dLPOD that does not contain the value 0 indicates a statistical significant difference between the two methods.

(i) 3M Molecular Detection heat block insert .—3M Food Safety. (j) 3M Molecular Detection cap/decap tool for reagent tubes .—3M Food Safety. (k) 3M Molecular Detection cap/decap tool for lysis tubes .—3M Food Safety. (l) Empty lysis tube rack .—3M Food Safety. (m) Empty reagent tube rack .—3M Food Safety. (n) ISO BPW .—3M Food Safety. Formulation equivalent to ISO 6579:2002 Annex B or 3M equivalent. (o) Disposable pipet .—Capable of 20 μL. (p) Multichannel (eight-channel) pipet .—Capable of 20 μL. (q) Sterile filter-tip 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 or 41.5 ± 1°C. (w) Refrigerator .—Capable of maintaining 2–8°C, for storing the 3M MDA components. (x) Computer .—Compatible with the 3M MDS instrument. (y) 3M Enviroswab .—Hydrated with Letheen Broth (3M Food Safety, Banglore, Australia). (z) 3M hydrated sponge stick with 10 mL D/E .—3M Food Safety.

C. General Instructions

( 1 ) Store the 3M MDA 2 – Salmonella 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.

03/10/2019

B ird et al .: J ournal of aoaC i nternational V ol . 99, n o . 4, 2016 985

9

Table 2016.01C. Comparative results for the detection of Salmonella at three inoculum levels in raw ground beef (73% lean) by the 3M MDA 2 – Salmonella method versus USDA/FSIS MLG reference method in a collaborative study

Candidate presumptive

Candidate confirmed

Candidate result

Reference method

Candidate versus reference

Matrix and inoculation level Laboratory

Statistic

POD CC

POD C

POD R

dLPOD C

dLPOD CP

N X POD CP

N X

N X

N X

Raw ground beef (73% lean), uninoculated control

1 2 3 4

12 0 0.00 12 12 0 0.00 12 12 2 0.17 12 12 2 0.17 NA a

0 0 2 0

0.00 12 0 0.00 12 0 0.00 0.00 0.00 0.00 12 0 0.00 12 0 0.00 0.00 0.00 0.17 12 2 0.17 12 1 0.08 0.09 0.00 0.00 NA 0 0.00 12 0 0.00 0.00 0.17

5 b NA NA NA NA NA NA NA NA NA NA NA NA NA NA 6 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 7 c NA NA NA NA NA NA NA NA NA NA NA NA NA NA 8 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 9 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 10 c NA NA NA NA NA NA NA NA NA NA NA NA NA NA 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 1 0.08 −0.08 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 15 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 16 12 0 0.00 12 1 0.08 12 0 0.00 12 0 0.00 0.00 −0.08

Estimate

All

156 4 0.03 156 3

0.02 156 2 0.01 156 2 0.01 0.00 0.01

LCL d UCL e

0.01 0.06 0.15 0.14 0.17 0.04 0.00 0.09 0.16 0.14 0.19

0.01 0.06 0.14 0.12 0.15 0.03 0.00 0.07 0.14 0.12 0.15

0.00 0.05 0.11 0.10 0.15 0.03 0.01 0.07 0.11 0.10 0.13

0.00 −0.03 −0.03 0.05 0.03 0.05

0.11 0.10 0.15 0.00 0.00 0.04 0.11 0.10 0.13

s r

LCL UCL

s L

LCL UCL

s R

UCL LCL

0.0315

0.0877

0.0184

0.5167

P T

Raw ground beef (73% lean), low inoculum level

1 2

12 6 0.50 12 12 5 0.42 12

6 5

0.50 12 6 0.50 12 7 0.58 −0.08 0.00 0.42 12 5 0.42 12 4 0.33 0.09 0.00

3 12 9 0.75 12 10 0.83 12 9 0.75 12 7 0.58 0.17 −0.08 4 12 5 0.42 NA 4 0.33 NA 4 0.33 12 3 0.25 0.08 0.08 5 b NA NA NA NA NA NA NA NA NA NA NA NA NA NA 6 12 6 0.50 12 6 0.50 12 6 0.50 12 7 0.58 −0.08 0.00 7 c NA NA NA NA NA NA NA NA NA NA NA NA NA NA 8 12 9 0.75 12 9 0.75 12 9 0.75 12 8 0.66 0.09 0.00 9 12 4 0.33 12 4 0.33 12 4 0.33 12 5 0.42 −0.08 0.00 10 c NA NA NA NA NA NA NA NA NA NA NA NA NA NA 11 12 6 0.50 12 6 0.50 12 6 0.50 12 3 0.25 0.25 0.00 12 12 8 0.66 12 8 0.66 12 8 0.66 12 7 0.58 0.08 0.00 13 12 8 0.66 12 8 0.66 12 8 0.66 12 8 0.66 0.00 0.00 14 12 3 0.25 12 3 0.25 12 3 0.25 12 6 0.50 −0.25 0.00 15 12 5 0.42 12 5 0.42 12 5 0.42 12 4 0.33 0.09 0.00 16 12 9 0.75 12 9 0.75 12 9 0.75 12 8 0.33 0.09 0.00

03/10/2019

986 B ird et al .: J ournal of aoaC i nternational V ol . 99, n o . 4, 2016

10

Table 2016.01C. ( continued )

Candidate presumptive

Candidate confirmed

Candidate result

Reference method

Candidate versus reference

Matrix and inoculation level Laboratory

Statistic

POD CC

POD C

POD R

dLPOD C

dLPOD CP

N X POD CP

N X

N X

N X

MPN, CFU/test portion

Estimate

0.67 0.53 0.83

All 156 83 0.53 156 83 0.53 156 82 0.53 156 77 0.49 0.03 0.00

LCL UCL

0.44 0.62 0.49 0.44 0.52 0.09 0.00 0.24 0.50 0.45 0.52

0.43 0.63 0.49 0.44 0.52 0.12 0.00 0.27 0.50 0.45 0.52

0.43 0.62 0.49 0.44 0.52 0.10 0.00 0.25 0.50 0.45 0.52

0.41 −0.10 −0.14 0.58 0.16 0.14

s r

0.49 0.45 0.52 0.06 0.00 0.22 0.50 0.45 0.52

LCL UCL

s L

LCL UCL

s R

UCL LCL

0.1725

0.0715

0.1272

0.2813

P T

Raw ground beef (73% lean), high inoculum level

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 b NA NA NA NA NA NA NA NA NA NA NA NA NA NA 6 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 7 c NA NA NA NA NA NA NA NA NA NA NA NA NA NA 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 c NA NA NA NA NA NA NA NA NA NA NA NA NA NA 11 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 12 12 11 0.92 12 11 0.92 12 11 0.92 12 12 1.00 −0.08 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 15 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 16 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00

MPN, CFU/test portion

Estimate

4.76 3.41 6.65

All 156 155 0.99 156 155 0.99 156 155 0.99 156 156 1.00 −0.01 0.00

LCL UCL

0.96 1.00 0.08 0.07 0.15 0.00 0.00 0.03 0.08 0.07 0.09

0.96 1.00 0.08 0.07 0.15 0.00 0.00 0.03 0.08 0.07 0.09

0.96 1.00 0.08 0.07 0.15 0.00 0.00 0.03 0.08 0.07 0.09

0.98 −0.04 −0.03 1.00 0.02 0.03

s r

0.00 0.00 0.15 0.00 0.00 0.15 0.00 0.00 0.21 1.00

LCL UCL

s L

LCL UCL

s R

UCL LCL

0.4395

0.4395

0.4395

P T

a NA = No data submitted by the collaborating laboratory. b Laboratory 5 was unable to conduct the analysis did not submit data for this matrix. c Laboratories 7 and 10 did not participate in the evaluation of this matrix.

d LCL = Lower confidence limit. e UCL = Upper confidence limit.

03/10/2019

B ird et al .: J ournal of aoaC i nternational V ol . 99, n o . 4, 2016 987

11

Table 2016.01D. Comparative results for the detection of Salmonella at three inoculum levels in peanut butter by the 3M MDA 2 – Salmonella method versus FDA BAM reference method in a collaborative study

Candidate presumptive

Candidate confirmed

Candidate result

Reference method

Candidate versus reference

Matrix and inoculation level Creamy peanut butter, uninoculated control

Statistic

POD CC

POD C

POD R

dLPOD C

dLPOD CP

Laboratory

N X POD CP N X

N X

N X

1 2

12 0 0.00 12 0 0.00 12 12 0 0.00 12 0 0.00 12 12 1 0.08 12 2 0.17 12 12 0 0.00 12 0 0.00 12

0 0.00 12 0 0.00 12 1 0.08 12 0 0.00 12

0 0.00 0.00 0 0.00 0.00

0.00 0.00

3 a

0 0.00 0.08 −0.09

4

0 0.00 0.00

0.00

5 b NA c NA NA NA NA NA NA NA NA NA NA NA NA NA 6 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 7 a 12 1 0.08 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.08 8 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 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 b NA NA NA NA NA NA NA NA NA NA NA NA NA NA 12 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 13 12 2 0.17 12 0 0.00 12 0 0.00 12 0 0.00 0.17 0.00 14 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 15 12 0 0.00 12 0 0.00 12 0 0.00 12 0 0.00 0.00 0.00 16 d NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Estimate

All

132 2 0.01 132 0 0.00 132

0 0.00 132 0 0.00 0.00

0.01

LCL e UCL f

0.00 0.05 0.11 0.10 0.16 0.04 0.01 0.08 0.12 0.11 0.15

0.00 0.03 0.00 0.00 0.16 0.00 0.00 0.16 0.00 0.00 0.23

0.00 0.03 0.00 0.00 0.16 0.00 0.00 0.16 0.00 0.00 0.23

0.00 −0.03 −0.01

0.03 0.03

0.05

0.00 0.00 0.16 0.00 0.00 0.16 0.00 0.00 0.23

s r

LCL UCL

s L

LCL UCL

s R

UCL LCL

0.0265

1.0000

1.0000

1.0000

P T

Creamy peanut butter, low inoculum level

0.00 0.00

1 2

12 6 0.50 12 6 0.50 12 12 8 0.66 12 8 0.66 12 12 5 0.42 12 6 0.50 12 12 6 0.50 12 6 0.50 12

6 0.50 12 8 0.66 12 5 0.42 12 6 0.50 12

6 0.50 0.00 6 0.50 0.16

3 a

5 0.42 0.00 −0.08

4

5 0.42 0.08

0.00

5 b NA NA NA NA NA NA NA NA NA NA NA NA NA NA 6 12 9 0.75 12 8 0.66 12 8 0.66 12 8 0.66 0.00 0.09 7 a 12 10 0.83 12 8 0.66 12 8 0.66 12 7 0.58 0.08 0.17 8 12 6 0.50 12 6 0.50 12 6 0.50 12 5 0.42 0.08 0.00 9 12 7 0.58 12 7 0.58 12 7 0.58 12 8 0.66 −0.08 0.00 10 12 7 0.58 12 7 0.58 12 7 0.58 12 5 0.42 0.16 0.00 11 b NA NA NA NA NA NA NA NA NA NA NA NA NA NA 12 12 5 0.42 12 5 0.42 12 5 0.42 12 6 0.50 −0.08 0.00 13 12 9 0.75 12 8 0.66 12 8 0.66 12 5 0.42 0.24 0.09 14 12 7 0.58 12 7 0.58 12 7 0.58 12 8 0.66 −0.08 0.00 15 12 8 0.66 12 8 0.66 12 8 0.66 12 6 0.50 0.16 0.00 16 d NA NA NA NA NA NA NA NA NA NA NA NA NA NA

03/10/2019

988 B ird et al .: J ournal of aoaC i nternational V ol . 99, n o . 4, 2016

12

Table 2016.01D. ( continued )

Candidate presumptive

Candidate confirmed

Candidate result

Reference method

Candidate versus reference

Matrix and inoculation level MPN, CFU/ test portion

Statistic

POD CC

POD C

POD R

dLPOD C

dLPOD CP

Laboratory

N X POD CP N X

N X

N X

Estimate 0.70

All 132 78 0.59 132 76 0.58 132 76 0.58 132 68 0.52 0.06 0.02

LCL UCL

0.55 0.89

0.50 0.68 0.50 0.45 0.52 0.00 0.00 0.16 0.50 0.45 0.52

0.49 0.66 0.51 0.45 0.52 0.00 0.00 0.13 0.51 0.46 0.52

0.49 0.66 0.51 0.45 0.52 0.00 0.00 0.13 0.51 0.46 0.52

0.43 −0.06 −0.11

0.61 0.18

0.14

0.51 0.45 0.52 0.00 0.00 0.15 0.51 0.46 0.52

s r

LCL UCL

s L

LCL UCL

s R

UCL LCL

0.8294

0.9590

0.9590

0.8762

P T

Creamy peanut butter, high inoculum level

1 2

12 12 1.00 12 12 1.00 12 12 12 1.00 12 12 1.00 12 12 12 1.00 12 12 1.00 12 12 12 1.00 12 12 1.00 12

12 1.00 12 12 1.00 0.00 12 1.00 12 12 1.00 0.00 12 1.00 12 12 1.00 0.00 12 1.00 12 12 1.00 0.00

0.00 0.00 0.00 0.00

3 a

4

5 b NA NA NA NA NA NA NA NA NA NA NA NA NA NA 6 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 7 a 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 11 b NA NA NA NA NA NA NA NA NA NA NA NA NA NA 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 15 12 12 1.00 12 12 1.00 12 12 1.00 12 12 1.00 0.00 0.00 16 d NA NA NA NA NA NA NA NA NA NA NA NA NA NA

MPN, CFU/ test portion

Estimate 5.35

All 132 132 1.00 132 132 1.00 132 132 1.00 132 132 1.00 0.00 0.00

LCL UCL

3.46 8.24

0.97 1.00 0.00 0.00 0.16 0.00 0.00 0.16 0.00 0.00 0.23

0.97 1.00 0.00 0.00 0.16 0.00 0.00 0.16 0.00 0.00 0.23

0.97 1.00 0.00 0.00 0.16 0.00 0.00 0.16 0.00 0.00 0.23

0.97 −0.03 −0.03

1.00 0.03

0.03

0.00 0.00 0.16 0.00 0.00 0.16 0.00 0.00 0.23

s r

LCL UCL

s L

LCL UCL

s R

UCL LCL

1.000

1.000

1.000

1.000

P T

a Results from Laboratories 3 and 7 were not used in statistical analysis due to a reported contamination of control samples and a deviation from the testing protocol, respectively. b Laboratories 5 and 11 were unable to conduct the analysis did not submit data for this matrix.

c NA = No data were submitted by the collaborating laboratory. d Laboratory 16 did not participate in the evaluation of this matrix.

e LCL = Lower confidence limit. f UCL = Upper confidence limit.

03/10/2019

B ird et al .: J ournal of aoaC i nternational V ol . 99, n o . 4, 2016 989

13

a false-negative result leading to the release of contaminated product. Another option is to transfer 10 μL NB enrichment into the LS tubes.

Store resealed pouches at 2–8°C for no longer than 1 month. Do not use 3M MDA 2 – Salmonella past the expiration date. ( 2 ) Follow all instructions carefully. Failure to do so may lead to inaccurate results.

( 2 ) The recommended size of the sampling area for verifying 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 and then up and down) or collect environmental samples after your current sampling protocol or according to the FDA BAM, USDA-FSIS MLG, or ISO 18593 (15) guidelines. (a) Prewarm ISO BPW enrichment medium to 41.5 ± 1°C depending on matrixes tested. See Table 2016.01E . (b) Aseptically combine the enrichment medium and sample. (c) Homogenize thoroughly by blending, stomaching, or hand-mixing for 2 ± 0.2 min. Incubate at 41.5 ± 1°C for 18–24 h. ( 1 ) Wet a cloth or paper towel with a 1–5% (v/v in water) household bleach solution and wipe the 3MMolecular Detection speed loader tray. ( 2 ) Rinse the 3MMolecular Detection speed loader tray with water. ( 3 ) Use a disposable towel to wipe the 3M Molecular Detection speed loader tray dry. ( 4 ) Ensure that the 3M Molecular Detection speed loader tray is dry before use. E. Preparation of the 3M Molecular Detection Speed Loader Tray

D. Sample Enrichment

(a) Foods.— ( 1 ) Allow ISO BPW enrichment medium to equilibrate to ambient laboratory temperature (20–25°C) or prewarm to 41.5 ± 1°C depending on matrixes tested. See Table 2016.01E for matrix-specific enrichment protocols. ( 2 ) Aseptically combine the enrichment medium and sample. For all meat and highly particulate samples, the use of filter bags is recommended. ( 3 ) Homogenize thoroughly for 2 ± 0.2 min. Incubate matrixes according to the instructions provided in Table 2016.01E . (b) Environmental samples (not analyzed for this collaborative study).— ( 1 ) Sample collection devices can be a sponge hydrated with a neutralizing solution to inactivate the effects of the sanitizers. 3M recommends the use of a biocide-free cellulose sponge. Neutralizing solution can be D/E neutralizing broth (NB) or LetheenBroth. It is recommended to sanitize the area after sampling. Caution : Should you select to use NB that contains aryl

sulfonate complex as the hydrating solution for the sponge, it is required to perform a 1:2 dilution (one part sample into one part sterile enrichment broth) of the enriched environmental sample before testing in order to reduce the risks associated with

Table 2016.01E. 3M MDA 2 – Salmonella enrichment protocols Sample matrix Sample size

Enrichment broth volume Enrichment temperature, ±1°C Enrichment time, h

Raw ground beef

25 g

225 mL ISO BPW (prewarmed) 975 mL ISO BPW (prewarmed) 225 mL ISO BPW (prewarmed) 975 mL ISO BPW (prewarmed)

41.5

10–24

325 g

Raw ground chicken

25 g

41.5

10–24 14–-24 18–24 18–24

325 g 325 g

Cooked breaded chicken

2925 mL ISO BPW 225 mL ISO BPW 1500 mL ISO BPW 225 mL ISO BPW

37 37

Dry dog food

25 g

375 g

Black pepper, raw whole shrimp, raw bagged spinach, pasteurized processed American cheese

25 g

37

18–-24

Chicken carcass rinse Chicken carcass sponge Instant nonfat dry milk

30 mL

30 mL ISO BPW (prewarmed) 50 mL ISO BPW (prewarmed)

41.5 41.5

18–24 18–24 20–24 24–28 18–24 18–24 18–24

1 sponge

25g 25g

225 mL ISO BPW 225 mL ISO BPW 900 mL ISO BPW 3375 mL ISO BPW 225 mL ISO BPW 3375 mL ISO BPW

37 37 37 37 37

Cocoa powder

Pasteurized liquid whole egg Spent sprout irrigation water

100 mL 375 mL

Creamy peanut butter

25 g

375 g

Environmental

Sealed concrete Stainless steel Sealed ceramic tile

1 sponge

225 mL ISO BPW (prewarmed) 10 mL ISO BPW (prewarmed) 50 mL ISO BPW (prewarmed)

41.5 41.5 41.5

18–24 18–24 18–24

1 swab

1 sponge

03/10/2019

990 B ird et al .: J ournal of aoaC i nternational V ol . 99, n o . 4, 2016

14

LS tubes in the 3M Molecular Detection heat block insert and heat for 15 ± 1 min. During heating, the LS solution will change from pink (cool) to yellow (hot). (e) 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 for a maximum of 10 min. The 3M Molecular Detection chill block insert, used at ambient temperature (20–25°C) without the 3M Molecular Detection chill block tray, should sit directly on the laboratory bench. When cool, the LS will revert to a pink color. (f) Remove the rack of LS tubes from the 3M Molecular Detection chill block insert. (a) One reagent tube is required for each sample and the NC. ( 1 ) Reagent tubes strips can be cut to the desired tube number. Select the number of individual reagent tubes or eight- tube strips needed. ( 2 ) Place reagent tubes in an empty rack. ( 3 ) Avoid disturbing the reagent pellets from the bottom of the tubes. (b) Select one reagent control (RC) tube and place in rack. (c) To avoid cross-contamination, decap one reagent tube strip at a time and use a new pipet tip for each transfer step. (d) Transfer lysate to reagent tubes and RC tube as described below: Note : Transfer each sample lysate into individual reagent tubes first , followed by the NC. Hydrate the RC tube last . ( 1 ) Use the 3M Molecular Detection cap/decap tool for reagent tubes to decap one reagent tube strip, one strip at a time. Discard cap. ( 2 ) Transfer 20 μL sample lysate from the upper one-half of the liquid (avoid precipitate) in the LS tube into the corresponding reagent tube. Dispense at an angle to avoid disturbing the pellets. Mix by gently pipetting up and down five times. ( 3 ) Repeat step I(d) (2) until individual sample lysate has been added to a corresponding reagent tube in the strip. ( 4 ) 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. ( 5 ) Repeat steps I(d) (1–4) , as needed, for the number of samples to be tested. ( 6 ) When all sample lysates have been transferred, repeat I(d) (1–4) to transfer 20 μL NC lysate into a reagent tube. ( 7 ) 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. (e) Load capped tubes into a clean and decontaminated 3M Molecular Detection speed loader tray. Close and latch the 3M Molecular Detection speed loader tray lid. See Figure 2016.01B . (f) Review and confirm the configured run in the 3M Molecular Detection software. (g) Click the Start button in the software and select instrument for use. The selected instrument’s lid automatically opens. (h) 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. I. Amplification

F. 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 or a 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. (a) Launch the 3M Molecular Detection software and log in. (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 MDS instrument must reach and maintain a 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. (a) Allow the LS tubes to warm up to room temperature by setting the rack on the laboratory bench for at least 2 h. Invert room-temperature capped lysis tubes to mix. Sample aliquots must be transferred to lysis tubes within 4 h of mixing. (b) Remove the enrichment broth from the incubator and gently agitate the contents. (c) One LS tube is required for each sample and the negative control (NC) sample. ( 1 ) LS tube strips can be cut to the desired LS tube numbers. Select the number of individual LS tubes or eight-tube strips needed. Place the LS tubes in an empty rack. ( 2 ) To avoid cross-contamination, decap one LS tubes strip at a time and use a new pipet tip for each transfer step. (d) Transfer enriched sample to LS tubes as described below: Note : Transfer each enriched sample into an individual LS tube first . Transfer the NC last . ( 1 ) Use the 3M Molecular Detection cap/decap tool for lysis tubes to decap one LS tube strip, one strip at a time. ( 2 ) Discard the LS tube cap. If lysate will be retained for retest, place the caps into a clean container for reapplication after lysis. ( 3 ) Transfer 20 μL sample into an LS tube. ( 4 ) Repeat step H(d) (2) until each individual sample has been added to a corresponding LS tube in the strip. See Figure 2016.01A . ( 5 ) Repeat steps H(d) (1-4) , as needed, for the number of samples to be tested. When all samples have been transferred, transfer 20 μL NC into an LS tube. Do not recap tubes. ( 6 ) Verify that the temperature of the 3M Molecular Detection heat block insert is at 100 ± 1°C. Place the rack of H. Lysis G. Preparation of the 3M Molecular Detection Instrument

03/10/2019

Made with FlippingBook Online newsletter