Total Collaborative Study Protocol_Solus One Salmonella v1 1

AOAC INTERNATIONAL 1  Official Methods of Analysis SM Program 2 

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Collaborative Study Protocol for the Solus One Salmonella for 4  the Detection of Salmonella spp. in Selected Foods and 5  Environmental Surfaces – OMACON‐2019‐6 6 

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For OMA First Action Status 10  September 2019  11  Version 1 

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Prepared for: 

Solus Scientific Solutions Ltd  9 Mansfield Network Centre 

Millennium Business Park, Concord Way 

Mansfield  NG19 7JZ 

United Kingdom 

Prepared by:  Maria Nelson 

2275 Research Blvd., Suite 300 

Rockville, MD 20850 

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Table of Contents

1  2  3  4  5  6  7  8  9 

1.0

Introduction 

1.1 1.2 1.3

Solus One  Salmonella

Summary of the Performance Tested Method SM  Validation 

Study Director 

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

Collaborators  Study Design 

Test Material Preparation and Distribution 

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Analysis and Confirmation 

Reporting Raw Data  Statistical Analysis 

Solus One  Salmonella  ‐ Method 

References  Appendices 

10.0

10.1 10.2 10.3 10.4 10.5

Instructions to Collaborators 

Study Materials  Flow Diagram 

Collaborator Information Sheet  Collaborator Comment Form 

11.0

Attachments 

1  2 

Solus One Salmonella 1 plate issue – Kit Insert  Solus One Salmonella 5 plate issue – Kit Insert 

3  PTM Report – Evaluation of the Solus One  Salmonella method for the Detection of  Salmonella  species in Select Food and Environmental Surfaces, AOAC Performance 

Tested Method SM  101801 

4  PTM Report – Method Modification to Extend the Matrix Claim of Solus One  Salmonella  method for the Detection of  Salmonella  species in Spices and Flavor blends, AOAC 

Performance Tested Method SM  101801  Solus One Salmonella – Data Report Form 

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1.0 1  2  The purpose of this collaborative study is to determine the performance of the candidate method  3  among collaborators for the detection of  Salmonella . Estimates of repeatability, reproducibility and  4  probability of detection (POD) will be evaluated. Collaborators will analyze one matrix at 3  5  contamination levels comparing the performance of the candidate method to appropriate reference  6  culture method. The Solus One  Salmonella  is the candidate method. The U.S. Food and Drug  7  Administration Bacteriological Analytical Manual  (FDA BAM) Chapter 5 (1) is the reference method for  8  this study. 9  10  1.1 Solus One  Salmonella Introduction  The Solus One  Salmonella method is an en zyme‐linked immunosorbent assay ( ELISA) designed  to detect  Salmonella  ( Salmonella enterica  subspecies enterica ,  salamae , arizonae , diarizonae ,  houtenae ,  indica and  Salmonella bongori ) in select foods and environmental samples following  enrichment containing a proprietary supplement. The method utilizes antibodies bound to the  wells of microplate strips that are specific to  Salmonella antigens and detects both motile and  non‐motile  Salmonella . Heat inactivated/lysed samples are added to the wells of the plate and  sample analysis is conducted following a manual or a fully automated protocol on the Dynex DS2  instrument. Results are based on optical density levels resulting from a color change reaction in  the wells. Presumptive positive or negative results can be obtained in as little as 22 h for most 

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food samples and 18 h for environmental samples. See Attachments 1 and 2. 

Summary of the Performance Tested Method SM  Validation 

1.2

The Solus One  Salmonella method was certified by the AOAC Research Institute in October,  2018 and designated Performance Tested Method SM  (PTM) 101801 for six food matrices; raw  beef trim (375 g), non‐fat dry milk powder (NFDM, 375 g), pasteurized liquid egg (100 g), raw  salmon (25 g), cheddar cheese (25 g), Romaine lettuce (25 g), and two environmental surfaces  (stainless steel and polystyrene). A method modification was approved in August 2019 to  include herbs, spices and flavorings; whole black peppercorns, paprika powder and cinnamon  powder (25 g sample size) plus honey mustard onion seasoning and flavored ranch seasoning 

(375 g sample size). See Attachments 3 and 4. 

1.3

Study Directors 

Ray Wakefield 

Solus Scientific Solutions, Ltd. 

9 Mansfield Networkcentre, Millennium Business Park 

Concorde Way, Mansfield 

Nottinghamshire NG19 7JZ, United Kingdom  Email:  ray.wakefield@solusscientific.com

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1  2  3  4  5  6  7 

Ben Bastin (Study contact) 

Q Laboratories 

1930 Radcliff Drive   Cincinnati, Ohio 45204  

Email: bbastin@qlaboratories.com

2.0 8  9  A total of 14–16 collaborators will be solicited to participate in this study. A minimum of 10 valid  10  laboratory data sets are required to successfully complete the study. Each collaborator will receive  11  instructions for performing the study (Appendix 10.1) and required materials prior to the start of the  12  study. Training on the DS2 Instrument and the Solus One  Salmonella method will be provided as  13  necessary.      16  17  A collaborative study will be conducted in accordance with the AOAC INTERNATIONAL Methods  18  Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces  19  (2). The Solus One  Salmonella method will be compared to the FDA BAM Chapter 5 reference method  20  for the detection of  Salmonella  in 375 g NFDM. A proprietary enrichment will be used for the Solus  21  method, so this study will be conducted as an unpaired study.    22  23  The NFDM will be screened for the presence of naturally contaminating  Salmonella and total aerobic  24  plate count ( note : for the FDA BAM method, if the level of background microflora is > 10 4  cfu/g, then the  25  tetrathionate (TT) broth must be incubated at 43 ± 0.2°C in a circulating, thermostatically‐controlled,  26  water bath, and if the level is ≤ 10 4  cfu/g, then TT should be incubated at 35 ± 2.0°C). Naturally  27  contaminated matrix is preferred if available. If naturally contaminated matrix is not available, artificially  28  contaminated matrix will be prepared. It is anticipated that artificial contamination will be required for  29  the NFDM. The matrix will be divided into 3 test materials. One test material remains non‐inoculated  30  and serves as the uncontaminated level, one test material is contaminated at a level that will produce a  31  reference method POD (POD R ) or candidate method POD (POD C ) in the range of 0.25–0.75  32  (approximately 0.2–2 CFU/test portion), and finally one test material is contaminated at a level to assure  33  a POD C of nearly 1.0 (approximately 10 CFU/test portion). Twelve replicate test portions per material  34  will be tested by the candidate method (36 test portions) and the reference method (36 test portions).    35  36  On the day that the analysis of the test samples is initiated, a 5‐tube 3‐level Most Probably Number  37  (MPN) estimation of contamination levels will be conducted using the FDA BAM reference method. The  38  MPN analysis scheme may also make use of the reference method replicates (see Appendix X‐A of the  39  AOAC Guidelines for details). The Least Cost Formulations, Ltd. (LCF) MPN Calculator‐Version 1.6 (3) will  40  be used to determine the MPN values and 95% confidence intervals. The MPN is reported as MPN/test  41  portion with 95% confidence intervals.  42 Collaborators  14  15  3.0 Study Design 

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1  2 

Table 1. Matrix Preparation 

Test  Portions/  Method 

Test  Portion  Codes  Random  number  100‐800 

Inoculation  Organism 

Reference  Method  FDA BAM  Ch. 5  

Matrix 

Target Levels   0 CFU/test portion 

12  12  12 

S. Senftenberg  ATCC TBD

NFDM 

0.2–2 CFU/ test portion  5–10 CFU/ test portion 

3  4 

4.0 5  6  To prepare artificially contaminated matrix, primary test materials will be inoculated with  Salmonella  so  7  that on the day of initiation of analysis  there is a low level (approx. 0.2–2 CFU/test portion) and a high  8  level (approximately 5–10 CFU/test portion). Note that one level of contamination must result in  9  fractional positive results. One test material will remain uninoculated to serve as the uncontaminated  10  control. The organizing laboratory will prepare and ship the test materials. Each sample will be divided  11  into test portions, randomized, and blind‐coded for analysis. Collaborators will complete and return the  12  data sheets to the study director following confirmation, within 2 weeks of the test initiation date.    Test Material Preparation and Distribution  4.1 NFDM will be obtained from a local retail outlet and screened for the presence of  Salmonella . Naturally contaminated test portions will be used if available at sufficient  levels. Alternatively, the test portions will be artificially contaminated to meet the  4.2 To artificially contaminate NFDM, a dried (lyophilized) inoculum will be used. The  inoculum will be added to a bulk test material and mixed by mechanical stirring or  rolling to achieve equal distribution of analyte throughout. Inoculated products will be  appropriate target levels.  

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stabilized for a minimum of 2 weeks at room temperature. 

4.3 The 375 g test portions for the Solus One  Salmonella method will be prepared by mixing  25 g of artificially contaminated material with 350 g of uncontaminated material for 

each contamination level. 

4.4

Test portions will be packaged in leak‐proof, insulated containers and shipped  (according to the Dangerous Goods Regulations IATA for Infectious Substances) by  overnight carrier to arrive the day before initiation of analysis . All test portions will be  shipped at ambient temperature (20–25°C). A temperature probe will be included with  each shipment to verify temperature of material upon receipt. Upon arrival, the test  portions will be stored at 20–25  C until they are analyzed. All participating collaborators 

will begin processing and analysis on the following Monday. 

Analysis and Confirmation 

5.0

5 

Each collaborator will receive a complete set of test materials (uncontaminated, low, and high) for each  1  method (candidate and reference) and will also receive one known uncontaminated sample to  2  determine the aerobic plate count (4). All participating collaborators will initiate sample analysis and  3  aerobic plate count for each matrix on the same day. One additional control (negative) test portion will  4  be provided to each collaborator for each matrix to determine the total plate count on the day of  5  analysis.  5.1 Each collaborator will receive one set of 375 g test portions (12 uncontaminated, 12  low, 12 high) to run on the Solus One Salmonella method, and one set of 25 g test  portions to run on the FDA BAM method. Each set will be blind coded so that the  6  7  8  9  5.2 The 375 g set of test portions will be enriched in Solus One  Salmonella 1125 mL Solus  One enrichment broth (BPW + supplement) at 41.5 ± 1°C for 21–22 h. Collaborators who  have the Dynex DS2 instrument in their laboratories will conduct the Solus One  Salmonella method using the Dynex instrument (automated procedure). Collaborators  who do not have the Dynex DS2 instrument in their laboratories will conduct the Solus  One  Salmonella method following the manual procedure. It is anticipated that there will  5.3 The 25 g set of test portions will be enriched in 225 mL brilliant green water contained  in sterile 500 mL Erlenmeyer flask or another appropriate container for 24 ± 2 h at 35°C. 5.4 After primary enrichment, ALL test portions (Solus One  Salmonella and FDA BAM) will  continue to selective enrichment. One (1) mL of each enriched portion will be  transferred to 10 mL TT broth and 0.1 mL will be transferred to 10 mL RV broth. Note:  BAM Method RV medium must be made from individual ingredients according to BAM  formulation.  Do not use commercial formulations. RV tubes will be incubated at 42 ±  0.2°C for 22–26 h using a circulating, thermostatically controlled water bath. TT tubes  will be incubated at 35 ± 2°C for 22–26 h. Secondary enrichments will be streaked to  bismuth sulfite (BS), xylose lysine desoxycholate (XLD), and Hektoen enteric (HE) agar  plates and incubated at 35°C for 22‐26 h. Isolated colonies will be transferred to TSI and  LIA slants and incubated 35 ± 2°C for 22‐26 h. Salmonella  colonies will be confirmed  using serological (Somatic O and poly H agglutination) and biochemical procedures  according to FDA BAM Ch. 5. The recovered isolates will be identified by the API20E  (Official Method 978.24 ), VITEK2 GN (Official Method 2011.17 ) or Bruker MALDI (OMA  5.5 In addition, all Solus One  Salmonella enriched test portions will be confirmed using an  alternative confirmation procedure. Each portion will be streaked directly from the  primary enrichment onto selective agar, as described by BAM Ch. 5. Confirmation will  continue through to identification from the selective agar as described in 5.4.    be an even mix of collaborators (automated and manual).   2017.09 ) depending on availability.   contamination level is unknown to the collaborator.  

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6.0

Reporting Raw Data 

6 

Each level of each matrix will be analyzed and reported separately. Data may be excluded due to an  1  assignable cause, provided there is sufficient justification. Such data will not be included in the statistical  2  analysis. All data, even if excluded from the statistical analysis, will be reported (see Appendix X‐B of the  3  AOAC Guidelines for raw data table format).    6  7  The statistical analysis will be performed in accordance with the AOAC Guidelines ( Appendix X‐F ) using  8  the Least Cost Formulations, Ltd., AOAC Binary Data Interlaboratory Study Workbook (5).    9  10  7.1 Estimate the repeatability standard deviation (s r ).  11  7.2 Estimate the reproducibility (s R ) by calculating the standard deviation of the laboratory  12  POD values (s POD ) and associated 95% confidence intervals.  13  7.3 Estimate the difference of cross‐laboratory POD (dLPOD) and associated 95% confidence  14  intervals. If the confidence interval of a dLPOD does not contain a zero, then the  15  difference is statistically significant.  16  7.3.1 dLPODc is the difference between the candidate and reference LPOD values.  17  7.3.2 dLPOD is the difference between the presumptive and confirmed LPOD values.  18  7.4 Graph POD R , LPOD C and dLPOD C by level with 95% confidence intervals. 19  7.5 Calculate false positive and false negative rates. 22  23  Solus One  Salmonella  for Detection of  Salmonella  in Selected Foods and Environmental Surfaces  24  25  (Applicable to the detection of  Salmonella  in raw beef trim (375 g), non‐fat dry milk powder (NFDM, 375  26  g), pasteurized liquid egg (100 g), raw salmon (25 g), cheddar cheese (25 g), Romaine lettuce (25 g),  27  whole black peppercorns, paprika powder and cinnamon powder (25 g sample size) plus honey mustard  28  onion seasoning and flavored ranch seasoning (375 g sample size) and stainless steel and polystyrene  29  surfaces)  32  Solus One Salmonella Method .— ( a )  Ensure that the heating block reaches a temperature of 85–100°C  33  and the sample is heated for 15–20 min thus ensuring the organisms are killed and the sample is safe to  34  handle.  35  ( b ) The Stop Solution contains sulfuric acid which is corrosive. Wash immediately with large  36  quantities of water if the solution comes into contact with skin or mucous membranes.  37  ( c )  All other Solus One  Salmonella  kit components are non‐hazardous. However, product usage  38  should follow good laboratory practices and all disposable materials must be discarded according to  39  appropriate waste procedures used in the laboratory.  40  ( d ) Protective clothing should be worn including laboratory coat, safety glasses, mask, and gloves  41  where appropriate. Avoid contact with the skin. 42 4  5  7.0 Statistical Analysis  20  21  8.0 Solus One  Salmonella – Method  30  31  Caution: 

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Dynex DS2 .— Improper use of the Dynex DS2 may cause personal injury or damage to the instrument.  1  For safe use, the Dynex DS2 must be operated only by qualified laboratory personnel who have been  2  appropriately trained. 3  Enrichment .— Salmonella  is a Biosafety Level 2 organism. Biological samples such as enrichments have  4  the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national  5  regulations on disposal of biological wastes. Wear appropriate protective equipment which includes but  6  is not limited to: protective eyewear, face shield, clothing/laboratory coat, and gloves. All work should  7  be conducted in properly equipped facilities utilizing the appropriate safety equipment (e.g., physical  8  contaminant devices). Individuals should be trained in accordance with applicable regulatory and  9  company/institution requirements before working with potentially infectious materials. All enrichment  10  broths should be sterilized following any culture based confirmatory steps through heat denaturation by  11  autoclaving at 121°C for 15 min.  16  proprietary media supplement – Solus One  Salmonella  supplement; for the rapid and specific detection  17  of  Salmonella  species in select foods and environmental samples. The modification described in this  18  report details the addition of five matrices: honey mustard onion seasoning and flavored ranch  19  seasoning (375 g sample size) plus cinnamon powder, paprika powder and whole black peppercorns (25  20  g sample size) in combination with a proprietary media – Solus modified Buffered Peptone Water  21  (mBPW).  22  Solus One  Salmonella  relies on antibodies attached to the wells of microplate strips by non‐covalent  23  biological interactions that are highly specific to Salmonella antigens. Samples are heat treated and an  24  aliquot is added to the antibody coated wells.  25  Salmonella  specific antigens present in the samples will bind immunologically to the antibody. After  26  washing to remove unbound material, an enzyme‐labelled antibody will bind to the captured proteins  27  and thus to the well. After a second wash step to remove any unbound enzyme‐antibody, the enzyme  28  substrate is added. The substrate reacts in the presence of the enzyme producing a blue color change in  29  the sample well. The substrate reaction is stopped after 30 minutes with the addition of dilute sulfuric  30  acid changing any blue color present in the wells to yellow (3). Optical densities resulting from this color  31  change are read within 10 minutes in a generic plate reader using a 450 nm filter (e.g. a microplate  32  reader or a Dynex DS2 instrument plate reader), where a result of an OD 450 < 0.200 is considered to be  33  negative for the target pathogen and OD 450 ≥ 0.200 is considered to be positive for the target pathogen.  12  13  14  15  A. Principle  Solus One  Salmonella  is an antibody‐based high sensitivity ELISA method paired with media and our 

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B. Apparatus 

( a )  Refrigerator .— capable of maintaining 2–8°C. 

( b )  Incubators .— capable of maintaining 37 ± 1°C and 41.5 ± 1°C.  ( c )  Circulating water bath .— capable of maintaining 42–50°C.  ( d )  Heating block or water bath .— capable of maintaining 85–100°C. 

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( e )  Autoclave for decontamination of samples . 

1  2  3  4  5 

( f )  Homogenizer 

(g)       Measuring cylinder .— capable of measuring 2 L.  ( h )  Vessel or container .— capable of storing 2 L. 

( i ) 

Filter laboratory blender bags . 

( j )  Sterile collection sponge and swabs for sampling environmental surfaces .  6  ( k )  Serological Pipette Bulbs (Automatic Pipette) .— For sampling and delivering of 1–10 mL.  7  ( l )  Serological pipettes .— Aerosol resistant.  8  ( m )  Precision pipettes .— For sampling and delivering of 100–300 µL, and 200–1,000 µL.  9  ( n )  Micropipette tips .— Aerosol resistant.  10  ( o )  Multi‐channel pipette .— Capable of delivering 100–300 µL.  11  ( p )  Microplate washer .  12  ( q )  Microplate reader with 450nm filter .  13  ( r )  Calibrated Thermometers .— Capable of measuring a temperature of 37 ± 1°C, 41.5 ± 1°C, and  14  85–100°C.  15  ( s )  Vortex mixer .— For swab homogenization.  16  ( t )  Calibrated Timer .  17  ( u )  Marker . 

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C.  Media and Reagents 

( a )  One or Five x 96 well microplates (in breakable strip format) .— Wells are coated with 

antibodies against  Salmonella  species. Store at 2–8°C. 

( b )  Negative Control (Green Label) .— 3 mL or 10 mL in working dilution. Contains diluent with 

preservative. Store at 2–8°C. 

( c )  Positive Control (Red Label) .— 3 mL or 10 mL in working dilution. Contains heat‐killed  26  Salmonella Agona in diluent with preservative. The positive control is black in color. Store at 2–8°C.  27  ( d )  Conjugate (Orange Label) .— 11 mL or 60 mL in working dilution. Contains horseradish  28  peroxidase‐antibody conjugate in diluent with preservative. Store at 2–8°C.  29  ( e )  Substrate (Blue Label) .— 11 mL or 60 mL in working dilution. Contains 3,3’,5,5’‐ 30  Tetramethylbenzidine (TMB), hydrogen peroxide and stabilizers. Note: Solution should be colorless.  31  Store at 2–8°C.  32  ( f )  Stop Solution (Silver Label) .— 11 mL or 60 mL in working dilution and contains 0.2M sulfuric  33  acid. Store at 2–8°C.  34  ( g )  Washing Buffer Concentrate (25x) .— 6 x 10 mL or 5 x 60 mL. Store at 2–8°C.  35  ( h )  Washing Buffer Activator .— 6 x 1 Sachet or 5 x 1 Sachet. Store at 2–8°C.  36  ( i )  Product Instructions . 

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D.  Additional supplies and reagents  

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( a )  Dehydrated Buffered Peptone Water (BPW) Medium according to ISO 6579, CAT No. MED017 .  1  ( b )       Dehydrated Solus modified Buffered Peptone Water (Solus mBPW), CAT No. MED038 .  2  ( c )  Solus One Salmonella Supplement , CAT No. SALSUPP‐22.5 & SALSUPP‐112 .  3  ( d )  Dynex Technologies DS2 Instrument (Dynex DS2) . — Dynex Technologies, Chantilly, VA.  4  ( e )  Computer with DS2 Software .  5  ( f ) Dynex Technologies 300 µL Sample Tips .  6  ( g ) Dynex Technologies 1300 µL Reagent Tips .  7  ( h )  Sample Tubes .— 12 mm external diameter and 75 mm height.  8  ( i ) Polypropylene Tubes .— 2 mL and 15 mL and 25 mL.  9  ( j ) Frit filters .— Optional for matrices with a lot of particulate , CAT No. FRI001.  10  ( k ) 70% v:v Ethanol .— For preparation of the Solus One  Salmonella Supplement.  11  ( l ) Deionized water . 12  ( m )  Sterile collection sponge for sampling environmental surfaces .— World Bioproducts EZ Reach  13  Sponge Sampler with Hi Cap Neutralizing Buffer, or equivalent. 14  ( n )  Sterile collection swab for sample environmental surfaces . 15  ( o )  Letheen Broth neutralizing buffer .

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E. General Preparation 

( a ) Use aseptic techniques. 

( b ) Use filter laboratory bags during enrichment to minimize particulates. 

( c )  Separate work areas for the following: media preparation, sample preparation, and pathogen 

detection. 

( d ) Clean the work stations with a disinfectant of choice before and after use. (Sodium 

24  hypochlorite solution, phenol solution, Quaternary ammonium solution, etc.)  25  ( e ) Do not reuse kit disposables.  26  ( g )  Change pipette tips in between samples. 

Pathogen Detection (Manual Method) 

( a ) Equilibrate test kit to room temperature (15–25°C) an hour before use. 

( b ) Ensure that the bench processing time of inoculated samples is kept to a minimum, to avoid 

extensive growth of competing non‐ Salmonella bacteria. 

( c )  Start heating block or heat water bath to 85–100°C prior to initiating testing.  ( d ) Allow heated samples to cool to room temperature (15–25°C). This may be accelerated by 

placing the test tubes in cold water for 5 min. 

( e ) Where appropriate frit filters will be used with denatured matrices that contain high 

37  concentrations of particulates thereby minimizing sample handling errors.  38  ( f )  Change pipette tips in between samples. 

39  40 

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Pathogen Detection (Automated Sample Preparation Method) 

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1  2  3  4  5 

( a ) Equilibrate test kit to room temperature (15–25°C) an hour before use.  ( b ) Start the Dynex DS2 and allow instrument to conduct the ‘Self‐Test’.  ( c )  Load required materials: pipette tips, reagents, washing buffer, etc. 

( d ) Empty waste. 

( e ) Start heating block or heat water to 85–100°C prior to initiating testing.  6  ( f )  Allow heated samples to cool to room temperature (15–25°C). This may be accelerated by  7  placing the test tubes in cold water for 5 min.  8  ( g )  Where appropriate use frit filters with denatured matrices that contain high concentrations of  9  particulates to minimize sample handling errors. 

10  11  12  13  14 

F. Sample Enrichment and Preparation

( a )  Raw Beef Trim (375 g test portions)

Pre‐warm 1,125 mL of Solus BPW (ISO) Medium supplemented with Solus  Salmonella  supplement  15  [4.44 mL Solus  Salmonella  supplement per 1,000 mL Solus BPW (ISO)] to 37 ± 1°C for at least 2 h prior to  16  its addition to a 375 g test portion in the sampling bag. Homogenize by hand massaging for 2 min ± 30 s  17  and incubate at 41.5 ± 1°C for 20–22 h.  18  ( b )  Pasteurized Liquid Egg (100 g test portions) 19  Pre‐warm 900 mL of Solus BPW (ISO) Medium supplemented with Solus  Salmonella  supplement  20  [4.44 mL Solus  Salmonella  supplement per 1,000 mL Solus BPW (ISO)] to 37 ± 1°C for at least 2 h prior to  21  its addition to a 100 g test portion in the sampling bag. Homogenize by hand massaging for 2 min ± 30 s  22  and incubate at 41.5 ± 1°C for 20–22 h.  23  ( c )  Raw Salmon (fillet), Cheddar Cheese (shredded) and Romaine Lettuce (bagged) (25 g test  24  portions) 25  Add 225 mL of Solus BPW (ISO) Medium supplemented with Solus  Salmonella  supplement [4.44 mL  26  Solus  Salmonella  supplement per 1,000 mL Solus BPW (ISO)] to a 25 g test portion in the sampling bag.  27  Homogenize for 2 min ± 30 s and incubate at 41.5 ± 1°C for 20–22 h.  28  ( d )  Paprika (powder) and Black Peppercorns (whole) (25 g test portions) 29  Add 225 mL of Solus mBPW Medium supplemented with Solus  Salmonella  supplement [2.22 mL  30  Solus  Salmonella  supplement per 1,000 mL Solus mBPW] to a 25 g test portion in the sampling bag.  31  Homogenize for 2 min ± 30 s and incubate at 41.5 ± 1°C for 20–24 h.  32  ( e )  Cinnamon (powder) (25 g test portions) 33  Add 1,225 mL of Solus mBPW Medium supplemented with Solus  Salmonella  supplement [2.22 mL  34  Solus  Salmonella  supplement per 1,000 mL Solus mBPW] to a 25 g test portion in the sampling bag.  35  Homogenize by hand massaging for 2 min ± 30 s and incubate at 41.5 ± 1°C for 20–24 h.  36  ( f )  Honey Mustard Onion Seasoning and Flavored Ranch Seasoning (375 g test portions) 37  Add 3,375 mL of Solus mBPW Medium supplemented with Solus  Salmonella  supplement [2.22 mL  38  Solus  Salmonella  supplement per 1,000 mL Solus mBPW] to a 375 g test portion in the sampling bag.  39  Homogenize by hand massaging for 2 min ± 30 s and incubate at 41.5 ± 1°C for 20–24 h.  40  ( g )  Non‐Fat Dry Milk Powder (375 g test portions) 41  Pre‐warm 1,125 mL of Solus BPW (ISO) Medium supplemented with Solus  Salmonella  supplement  42

11 

[4.44 mL Solus  Salmonella  supplement per 1,000 mL Solus BPW (ISO)] to 37 ± 1°C for at least 2 h prior to  1  its addition to a 375g test portion in the sampling bag. Homogenize by hand massaging for 2 min ± 30 s  2  and incubate at 41.5 ± 1°C for 21–22 h.  3  ( h )  Stainless Steel environmental surface 4  For 4” x 4” surface area, sample surface with sterile sampling sponge device (e.g. World Bioproducts  5  EZ Reach Sponge Sampler with HiCap neutralizing broth or Hygiena equivalent). Follow sampling device  6  manufacturers’ instructions for correct use, storage and transport. Pre‐warm 100 mL of Solus BPW (ISO)  7  Medium supplemented with Solus  Salmonella  supplement [4.44 mL Solus  Salmonella  supplement per  8  1,000 mL Solus BPW (ISO) to 37 ± 1°C for at least 2 h prior to its addition to the sampled sponge in the  9  sampling bag. Homogenize by hand massaging for 2 min ± 30 s and incubate at 41.5 ± 1°C for 16–20 h.  10  ( i )  Plastic environmental surface 11  For 1” x 1” surface area, sample surface with sterile sampling swab (e.g. sponge or foam pre‐ 12  moistened with Letheen broth). Follow sampling device manufacturers’ instructions for correct use,  13  storage and transport. Pre‐warm 10 mL of Solus BPW (ISO) Medium supplemented with Solus  14  Salmonella  supplement [4.44 mL Solus  Salmonella  supplement per 1,000 mL Solus BPW (ISO)] to 37 ±  15  1°C for at least 2 h prior to its addition to the sampled swab in the sampling tube. Homogenize by vortex  16  mixing for 2 min and incubated at 41.5 ± 1°C for 16–20 h.  17  ( j )  Following the incubation, gently agitate each bag or tube and transfer a 1 mL aliquot to a  18  polypropylene test tube. Heat tubes at a temperature of 85–100°C for 15 – 20 min.  19  ( k )  After heat treatment, cool the samples to room temperature (15–25°C). This may be  20  accelerated by placing the test tubes in cold water for 5 min.  21  ( l )  Where appropriate use frit filters with denatured matrices that contain high concentrations of  22  particulates to minimize sample handling errors.  23  ( m ) Keep the remaining non‐heat‐treated samples for verification until ELISA results are obtained.  24  These samples can be stored at 41.5 ± 1°C if the ELISA test is to be carried out within 2 h or at 2–8°C for  25  up to 72 h if not.   30  room temperature (15–25°C). Determine the number of wells required for the test. Take the required  31  number of strips from the pouch and fit them to the framer provided. Note: Unused strips should be  32  returned to the pouch and stored at 2–8°C.  33  ( b ) Prepare the Washing Buffer by adding the contents of a single Washing Buffer Activator  34  sachet and Washing Buffer bottle (60 mL) to 1440 mL or a single Washing Buffer Activator sachet and  35  Washing Buffer bottle (10 mL) to 240 mL of deionized water to prepare the washing buffer at assay  36  concentration. Mix until the activator has fully dissolved.  37  ( c )  Leave the first well in the strip empty to serve as a “blank” for measuring the absorbance of  38  the substrate.  39  ( d ) Aliquot 0.1 mL of the Negative Control in the second well and 0.1 mL of the Positive Control in  40  the third well.  41  ( e ) Add 0.1 mL of each heated sample to each consecutive well in the strip. If there are wells left  42 26  27  28  29  G. Solus One Salmonella (Manual Method)  ( a ) Remove the kit from storage at 2–8°C an hour before use to allow the components to reach 

12 

over at the end of a test strip the Positive or Negative Controls may be repeated.  1  ( f )  Incubate the plate (containing the strips) at 37 ± 1°C for 30 ± 5 min.  2  ( g )  Following incubation, aspirate the contents of the wells, removing as much of the liquid as  3  possible. Wash the wells 5–7 times with Washing Buffer ensuring completed filling and emptying of the  4  wells through each wash cycle. Note: The washing technique is critical to assay performance; hence it is  5  recommended to use a microplate washer.    6  ( h ) After completion of the washing steps, add 0.1 mL of Conjugate to each well, except for the  7  blank. Upon completion of the addition of the Conjugate, incubate the plate (containing the strips) at 37  8  ± 1°C for 30 ± 5 min.  9  ( i )  Repeat the aspiration and wash cycles.  10  ( j )  After completion of the second aspiration and washing steps, add 0.1 mL of TMB Substrate to  11  each well, including the blank. Upon completion of the addition of the TMB Substrate, incubate the  12  plate (containing the strips) for 30 ± 5 min at room temperature (15–25°C).  13  ( k )  After incubation, stop the reaction by adding 0.1 mL of Stop Solution to all wells, including the  14  “blank” well. Note: The Stop Solution will cause any blue color in wells to change to yellow.  15  ( l )  Where applicable read the optical densities within 10 min in a microplate reader using a 450  16  nm filter (do not use reference filter), or on the Dynex DS2 instrument using the “Plate Read Only”  17  setting. Inspect the wells before reading for air bubbles and if present burst with a sterile needle. Zero  18  the reader against the “blank” well before the other wells are read.  19  20  H. Solus One Salmonella (Automated Sample Preparation Method) 

21  22  23  24 

( a ) Prior to analysis, bring the entire contents of the kit to room temperature (15–25°C) by placing 

the kit on a sanitized bench top for 1 h prior to use. 

( b ) Determine the number of wells required for the test. Take the required number of strips from  25  the pouch and fit them to the frame provided. Note: Unused strips should be returned to the pouch and  26  stored at 2–8°C. 

27  28  29  30  31  32  33  34  35  36  37  38  39  40  41 

I. Analysis 

( a )  Prepare equipment

1) Turn on power to the Dynex DS2 instrument, followed by the power to the computer.  Open the Matrix software on the computer and enter login information. The Dynex DS2 

instrument will perform a self‐diagnostic check. 

( b )  Create a Run File 

1) In the DS‐ Matrix software, select “File”, “Worklist Editor”. Then select “New Sample 

Batch”. 

2) Enter your sample batch ID as required.  3) Enter the total number of samples in the batch. 

4) Sample ID may then be typed into the empty positions in the rack or assigned 

automatically using an onboard barcode scanner. 

42

5) If using an onboard barcode scanner slide the sample tube racks onto the machine and 

13 

1  2  3  4  5  6  7  8  9 

click next. 

6) Click on the Solus One  Salmonella assay. Then click the “Add Assay” button.  

7) Select “Done” when assay selection is completed.  8) Select the “Accept” button to start the run.  

9) After the timeline has been accepted, loading of sample tube racks, reagents and 

consumables is initiated. 

( c )

Loading the Dynex DS2 Instrument 

1) If not already onboard, load sample test tube racks into the slots.   2) Remove the lid from the pipette boxes and load into the instrument.  

10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29 

3) Load the Conjugate, TMB Substrate, and Stop Solution into the correct locations specified 

by the instrument.  

4) Load the Negative and Positive Controls in each position specified by the instrument.  5) Prepare and dispense the correct amount of Activated Washing Buffer. 

6) Verify all disposable containers do not need emptying. 

( d )  Remove Samples 

1) When the run is completed, open the lid to the Dynex DS2 instrument and remove the  loading trays, with the sample test tubes, by pulling the loading trays out away from the  instrument. Discard sample test tubes in the proper location according to laboratory 

procedures. 

2) Discard the reagent tubes, negative controls, and positive controls in the proper location  according to laboratory procedures. Note: Reagents may be capped and refrigerated at 2–

8°C with the rest of the remaining kit. 

J. Test Result Report 

( a ) To view a report on a completed run, select the desired result file in the “Recent Test” Box, 

and select the “Report” button. 

( b ) The report can be viewed and printed from the report display screen. 

( c )  The results are displayed in a 96 well format with each individual optical density reading.  ( d ) A secondary file which can be used to export the results into a LIMS system is created in a  30  folder on the desk top called “ELISA Export Results”, this is in a CSV (comma separated values) file  31  format. 

32  33  34  35 

K. Interpretation 

The presence or absence of the pathogen is expressed as optical density (OD 450 36  using the Dynex DS2 instrument plate reader or a microplate reader with a 450 nm filter where a Dynex  37  DS2 instrument is unavailable. For control assay acceptance criteria, a result of an OD 450 < 0.100 is valid  38  for a Negative Control, whereas a result of an OD 450 > 0.500 is valid for a Positive Control. A result of an  39  OD 450 < 0.200 is considered to be negative for  Salmonella . A result of an OD 450 ≥ 0.200 is considered  40  presumptive positive for  Salmonella .  41  42 ) measurements 

14 

L. Confirmation  1  2  All samples are confirmed according to the appropriate reference methods specified for the matrix  3  and analyte. 

4  5  6  7  8  9 

9.0

References 

1. U.S. Food and Drug Administration Bacteriological Analytical Manual , Chapter 5 (2018),  https://www.fda.gov/food/laboratory‐methods‐food/bacteriological‐analytical‐manual‐

10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41

bam‐chapter‐5‐salmonella

2. Official Methods of Analysis  (2019), 21 st  Edition, Appendix J, AOAC INTERNATIONAL, 

Gaithersburg, MD, 

http://members.aoac.org/aoac_prod_imis/AOAC_Docs/StandardsDevelopment/eoma_

appendix_j.pdf

3.

Least Cost Formulations, Ltd., MPN Calculator‐Version 1.6 

4. U.S. Food and Drug Administration Bacteriological Analytical Manual , Chapter 3 (2001) 

https://www.fda.gov/food/laboratory‐methods‐food/bam‐aerobic‐plate‐count

5. Least Cost Formulations, Ltd., AOAC Binary Data Interlaboratory Study Workbook (2013) 

AOAC Interlaboratory Study Workbook ‐ Binary Data (Excel file .xls)

15 

10.0

Appendices 

1  2  3  4 

Appendix 10.1 Instructions to Collaborators 

General Instructions about Collaborative Studies  5  6  The purpose of this document is to provide detailed instructions for performing the collaborative study  7  for the Solus One Salmonella .    8  9  The trial will be conducted by Q Laboratories, on behalf of Solus Scientific. Ben Bastin of Q Laboratories  10 will be the main contact. Q Laboratories will be responsible for providing the test portions, clarifying  11  procedures, collating the results. Should any questions arise before or during the study, please direct  12  them immediately to the attention of one of Ben Bastin:  13   14 Ben Bastin  15  Q Laboratories  16  Email: BBastin@QLaboratories.com 17  513‐471‐1300 – work  18  513‐518‐8450 – cell   19   20 Important Information  21 22 1.  Read the methods carefully. If you have any questions, contact the Study Director.  23   24 2.  It is advised to make at least one practice run before the trial using your own materials so that you  25 can minimize errors in manipulations.  Check that all pipettes, equipment, and supplies are on hand.  26  27 3.  Make the determination on the specified date. Store the test portions according to the instructions.   28  It is essential for the validity of the trial that all collaborators commence the analysis of each test portion  29  on the designated day. Immediately upon receiving each shipment, confirm the contents with the Study  30  Director by emailing the form provided in the shipment.  31   32 4.  THIS IS A STUDY OF THE METHOD, NOT OF THE LABORATORY. THE METHOD MUST BE FOLLOWED AS  33 CLOSELY AS PRACTICABLE, AND ANY DEVIATIONS FROM THE METHOD AS DESCRIBED, NO MATTER HOW  34  TRIVIAL THEY MAY SEEM, MUST BE NOTED ON THE REPORT FORM.  35   36 5.  Report all of your results as soon as analyses are completed. Do not do more or less than indicated in  37  the instructions. For example, do not do duplicate analysis and report the best or average result. More  38  or fewer results complicate the statistical analyses and may invalidate your results. Data sheets are  39  provided with these instructions and indicate which results are to be reported. Please include any  40  criticisms, suggested improvements, or general comments about the products on the Collaborators’  41  Comments Form provided. Any results that were derived from modified protocols should be included  42

16 

but must be separated from the main report. Results and comments should be returned to the Study  1  Director immediately upon completion of each portion of the study. 

2  3 

Shipment Schedule  4  5  Test portions will be shipped by overnight express courier to arrive the day before initiation of analysis.   6  If the test portions do not arrive by the normal delivery time one day prior to analysis, please contact  7  the Study Director. Analysis of test portions must be initiated on the day scheduled by the Study  8  Director.   9  10  Test Portion Receipt  11  12  Note condition of package on the data sheets and notify the Study Director if any packages appear to  13  have been compromised or if the food product does not arrive in the appropriate condition. Dry  14  products should be stored at room temperature until the day of analysis.  Solus One Salmonella  19  20  The Solus One  Salmonella  kit components are non‐hazardous. However, product usage should follow  21  good laboratory practices and all disposable materials must be discarded according to appropriate waste  22  procedures used in the laboratory.  25  26  This product is not hazardous. However, product usage should follow good laboratory practices and all  27  disposable materials must be discarded according to appropriate waste procedures used in the  28  laboratory.  31  32  Salmonella  is a Biosafety Level 2 organism. Biological samples such as enrichments have the potential to  33  transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations on  34  disposal of biological wastes. Wear appropriate protective equipment which includes but is not limited  35  to protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in  36  properly equipped facilities utilizing the appropriate safety equipment (for example, physical  37  containment devices). Individuals should be trained in accordance with applicable regulatory and  38  company/institution requirements before working with potentially infectious materials. All enrichment  39  broths should be sterilized following any culture based confirmatory steps.   23  24  Matrix Control Kit  29  30  Enrichment  15  16  17  18  Safety Precautions 

40  41 

Test Portion Analysis

42

17 

1  Collaborators will be provided with enough test material to test each method (Solus One  Salmonella and  2  FDA BAM Ch. 5). Collaborators will receive two sets of test portions: 36 x 375 g (Solus One  Salmonella )  3  and 36 x 25 g (FDA BAM). One temperature control sample will also be included in the shipment to  4  document the temperature of the sample upon receipt. One additional 30 g control (uncontaminated)  5  test portion will be provided to each collaborator to determine the total plate count on the day of test  6  initiation. Record all results on the data sheets provided. See Attachment 5 for data sheets. Please  7  review both methods carefully before initiating analysis. 

8  9  10 

Reference  Pre‐enrichment 

Food Type 

Reference  FDA BAM a

Solus One  Salmonella

NFDM  BPW + supplement a https://www.fda.gov/food/laboratory‐methods‐food/bacteriological‐analytical‐manual‐bam‐ 12  13  14  A brief description of the methods is listed here. Follow the Solus One  Salmonella  kit insert and FDA  15  BAM Ch. 5 instructions when conducting the experiments.   Brilliant Green water  11  chapter‐5‐salmonella

16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36 

1. 

Solus One  Salmonella

1.1  To each 375 g NFDM portion, homogenize (2 min) in 1125 mL of supplemented BPW 

(pre‐warmed to 37°C) and incubate portions at 41.5 ± 1°C for 21–22 h.

1.2  When incubation is complete, carefully remove a 1 mL aliquot, avoiding particulate and  fatty matter. Heat aliquot to 85–100°C for 15–20 min. Allow to cool to room  temperature (20–25°C), or place test tubes in cold water for 5 min. Keep the unheated 

samples for confirmation. Samples can be stored up to 72 h at 2–8°C.

1.3  Prior to analysis, bring the entire contents of the kit to room temperature (15–25°C) by 

placing the kit on a sanitized bench top for 1 h prior to use. 

1.4  Determine the number of wells required for the test. Take the required number of strips  from the pouch and fit them to the frame provided. Note: Unused strips should be 

returned to the pouch and stored at 2–8°C. 

1.5  Dynex DS2 Automated procedure : Turn on power to the Dynex DS2 instrument,  followed by the power to the computer. Open the Matrix software on the computer and  enter login information. The Dynex DS2 instrument will perform a self‐diagnostic check.  1.6  In the DS‐ Matrix software, select “File”, “Worklist Editor”. Then select “New Sample 

Batch”. 

1.7  1.8  1.9 

Enter your sample batch ID as required.  Enter the total number of samples in the batch. 

37

Sample ID may then be typed into the empty positions in the rack or assigned 

18 

1  2  3  4  5  6  7  8  9 

automatically using an onboard barcode scanner. 

1.10  If using an onboard barcode scanner slide the sample tube racks onto the machine and 

click next. 

1.11  Click on the Solus One  Salmonella assay. Then click the “Add Assay” button.  

1.12  Select “Done” when assay selection is completed.  1.13  Select the “Accept” button to start the run.  

1.14  After the timeline has been accepted, loading of sample tube racks, reagents and 

consumables is initiated. 

1.15  If not already onboard, load sample test tube racks into the slots.   1.16  Remove the lid from the pipette boxes and load into the instrument.   1.17  Load the Conjugate, TMB Substrate, and Stop Solution into the correct locations 

10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41 

specified by the instrument.  

1.18  Load the Negative and Positive Controls in each position specified by the instrument.  1.19  Prepare and dispense the correct amount of Activated Washing Buffer. 

1.20  Verify all disposable containers do not need emptying. 

1.21  When the run is completed, open the lid to the Dynex DS2 instrument and remove the  loading trays, with the sample test tubes, by pulling the loading trays out away from the  instrument. Discard sample test tubes in the proper location according to laboratory 

procedures. 

1.22  Discard the reagent tubes, negative controls, and positive controls in the proper location  according to laboratory procedures. Note: Reagents may be capped and refrigerated at 

2–8°C with the rest of the remaining kit. 

1.23  To view a report on a completed run, select the desired result file in the “Recent Test” 

Box, and select the “Report” button. 

1.24  The report can be viewed and printed from the report display screen.  1.25  The results are displayed in a 96 well format with each individual optical density 

reading. 

1.26  A secondary file which can be used to export the results into a LIMS system is created in  a folder on the desktop called “ELISA Export Results”, this is in a CSV (comma separated 

values) file format. 

1.27  Interpretation: The presence or absence of the pathogen is expressed as optical density  (OD 450 ) measurements using the Dynex DS2 instrument plate reader or a microplate  reader with a 450 nm filter where a Dynex DS2 instrument is unavailable. For control 

assay acceptance criteria, a result of an OD 450

< 0.100 is valid for a Negative Control, 

> 0.500 is valid for a Positive Control. A result of an OD 450 < 

whereas a result of an OD 450

0.200 is negative for  Salmonella . A result of an OD 450

≥ 0.200 is considered presumptive 

positive for  Salmonella . 

1.28  Manual Procedure : Remove the kit from storage at 2–8°C an hour before use to allow  the components to reach room temperature (15–25°C). Determine the number of wells  required for the test. Take the required number of strips from the pouch and fit them to  the framer provided. Note: Unused strips should be returned to the pouch and stored at 

42

2–8°C 

19