PROCEDURE Molecular Detection Assay 2 – E. coli O157 (including H7) ANALYSIS of E. coli BY AOAC 2017.01

1. APPLICATION OF METHOD 3M Molecular Detection Assay 2- E. coli O157:H7 (including H7) is used with the 3M Molecular Detection System for the rapid and specific detection of E. coli O157:H7 (including H7) in enriched food, feed and food process environmental samples.

2. APPLICABLE MATRICES This method may be used for the rapid and specific detection of E. coli O157:H7 in enriched food, feed, and food process environmental samples.

3. METHOD DETECTION LIMITS The method LOD is defined as the lowest concentration where reliable analytical results can be obtained. This may vary with different species, strains, samples, and methods. For the 3M method, this has been demonstrated to be 1-5 CFU/sample.

4. SCOPE AND APPLICATION This method may be used for the rapid and specific detection of E. coli O157:H7 in enriched food and environmental samples.

5. SUMMARY OF METHOD The 3M Molecular Detection Assays use loop-mediated isothermal amplification to rapidly amplify nucleic acid sequences with high specificity and sensitivity, combined with bioluminescence to detect the amplification. Presumptive positive results are reported in real-time while negative results are displayed after the assay is completed.

6. DEFINITIONS 6.1. Negative control test: A representative clean matrix that is prepared and analyzed like all other samples in the perspective batch. The negative control test is used to demonstrate that the system is free of contamination and is analyzed for each new lot of reagents. For the MDS, it is a sterile enrichment medium, e.g., Buffered Peptone Water (BPW). Do not use water as a Negative Control.

6.2. Sample: A representative portion of any material collected from any source for which determination of contamination level is requested or required.

6.3. Matrix: A type of product with intrinsic properties such as composition and process. Differences between matrices may be as simple as the effects caused by differences in their processing or presentation for example, raw vs. pasteurized; fresh vs. dried, etc.

1. EQUIPMENT & SUPPLIES • Lysis Solution (LS) tubes • Lysis Tube rack • E. coli O157 (including H7) Reagent tubes • Reagent Tube rack • Extra caps • Incubator capable of maintaining 37±1°C • Reagent Control (RC) • 3M Molecular Detection Heat Block Insert • Pipette tips • Pipette • 3M Molecular Detection Assay Matrix Control kit • 3M Molecular Detection Speed Loader Tray • 3M Molecular Detection Chill Block Insert • 3M Molecular Detection Heat Block Insert • 3M Molecular Detection Cap/Decap Tool-Lysis • 3M Molecular Detection Cap/Decap Tool-Reagent • 3M Molecular Detection Software Disc • 3M Molecular Detection Instrument MDS 100 • Thermometer – partial immersion or digital thermocouple thermometer. Do not use a total immersion thermometer • External Power Supply • External Power Supply Cord(s)

2. REAGENTS & STANDARDS • Buffered Peptone Water ISO • Negative Control – sterile enrichment medium e.g., Buffered Peptone Water. Do not use water.

3. INTERFERENCES 9.1. Interferences may be caused by contamination of the workbench. To resolve this issue, disinfect the workbench with 10% bleach solution, and use sterile, aerosol (filtered), molecular biology grade pipette tips.

9.2. Never open tubes post amplification.

9.3. Store the 3M Molecular Detection Assay 2- E. coli O157:H7 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.

9.4. After opening, unused reagent tubes should always be stored in the re-sealable pouch with the desiccant inside to maintain stability of the lyophilized reagents.

9.5. Store resealed pouches at 2-8°C for no longer than 60 days.

9.6. Do not use 3M Molecular Detection Assay 2- E. coli O157:H7 past the expiration date.

9.7. Do not exceed the recommended temperature setting on heater.

9.8. Do not exceed the recommended heating time.

9.9. Use an appropriate, calibrated thermometer to verify the 3M Molecular Detection Heat Block Insert temperature (e.g., a partial immersion thermometer or digital thermocouple thermometer, not a total immersion thermometer.)

1. SAMPLE COLLECTION The client or the client’s representative submits samples and a completed Chain of Custody (COC) form to the field laboratory. Once received, the samples are inspected for labeling accuracy and any abnormalities. The sample containers are labeled with the appropriate SDG ID, date and time received and contents, and the samples are refrigerated at 4oC (±2oC) until the time of analysis.

2. PROCEDURE
   11.1. Sample Enrichment Foods: 11.1.1. Pre-warm BPW ISO enrichment medium to 41.5±1°C depending upon matrices tested. See Table 2. 11.1.2. Aseptically combine the enrichment medium and sample. For all meat and highly particulate samples, the use of filter bags is recommended. 11.1.3. Homogenize all matrices except leafy produce and fruit thoroughly by blending, stomaching, or hand mixing for 2±0.2 minutes. Incubate as outlined in the appropriate protocol table (See Table 2)

11.2. Preparation of the 3M Molecular Detection Speed Loader Tray: 11.2.1. Wet a cloth with a 1-5% (v:v in water) household bleach solution and wipe the 3M Molecular Detection Speed Loader Tray. 11.2.2. Rinse the 3M Molecular Detection Speed Loader Tray with water. 11.2.3. Use a disposable towel to wipe the 3M Molecular Detection Speed Loader Tray dry. 11.2.4. Ensure the 3M Molecular Speed Loader Tray is dry before use.

11.3. Preparation of the 3M Molecular Detection Chill Block Insert: Place the 3M Molecular Detection Chill Block directly on the laboratory bench; (the 3M Molecular Detection Chill Block Tray is not used). Use the chill block at ambient temperature (20-25°C).

11.4. Preparation of the 3M Molecular Detection Heat Block Insert: 11.4.1. Place the 3M Molecular Detection Heat Block Insert in a dry double 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. Depending on the heater unit, allow approximately 30 minutes for the 3M Molecular Detection Heat Block Insert to reach temperature. Using an appropriate, calibrated thermometer, (e.g., a partial immersion thermometer or 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.

11.5. Preparation of the 3M Molecular Detection Instrument 11.5.1. Launch the 3M Molecular Detection Software and log in. 11.5.2. Turn on the 3M Molecular Detection Instrument. 11.5.3. Create or edit a run with data for each sample. Refer to the 3M Molecular Detection System User Manual for details. NOTE: The 3M Molecular Detection Instrument must reach and maintain temperature of 60°C before inserting the 3M Molecular Detection Speed Loader Tray with reaction tubes. The heating step takes approximately 20 minutes 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.

11.6. Lysis 11.6.1. Allow the lysis solution (LS) tubes to warm up by setting the rack at room temperature (20-25°C) overnight (16-18 hours). Alternatives to equilibrate the LS tubes to room temperature are to set the LS tubes on the laboratory bench for at least 2 hours, incubate the LS tubes in a 37±1°C incubator for 1 hour or place them in a dry double heater for 30 seconds at 100°C. 11.6.2. Invert the capped tubes to mix. Proceed to next step within 4 hours. 11.6.3. Remove the enrichment broth from the incubator. 11.6.4. One LS tube is required for each sample and the Negative Control (NC) (sterile enrichment medium) sample. 11.6.4.1. LS tube strips can be cut to desired LS tube number. Select the number of individual LS tubes or 8-tube strips needed. Place the LS tubes in an empty rack. 11.6.4.2. To avoid cross-contamination, decap one LS tubes strip at a time and use a new pipette tip for each transfer step. 11.6.4.3. Transfer enriched sample to LS tubes as described below: Transfer each enriched sample into an individual LS tube first. Transfer the NC last. 11.6.4.4. Use the 3M Molecular Detection Cap/Decap Tool-Lysis to decap one LS tube strip-one strip at a time. 11.6.4.5. Discard the LS tube cap-if lysate will be retained for retest, place the caps in a clean container for re-application after lysis. For processing of retained lysate, see Appendix A. 11.6.4.6. Transfer 20 µL of sample into a LS tube unless otherwise indicated in Protocol Table 2. e.g. Raw dairy products use 10 µL. 11.6.5. Repeat step 11.6.4.2 until each individual sample has been added to a corresponding LS tube in the strip.

11.6.6. Repeat step 11.6.4.1 to 11.6.4.6 as needed, for the number of samples to be tested. 11.6.7. When all samples have been transferred, transfer 20µL of NC (sterile enrichment medium, e.g., BPW) into a LS tube. Do not use water as a NC. 11.6.8. Verify that the temperature of the 3M Molecular Detection Heat Block Insert is at 100±1°C. 11.6.9. Place the uncovered rack of LS tubes in the 3M Molecular Detection Heat Block Insert and heat for 15±1 minutes. During heating, the LS solution will change from pink (cool) to yellow (hot). Samples that have not been properly heat treated during the assay lysis step may be considered a potential biohazard and should NOT be inserted into the 3M Molecular Detection Instrument. 11.6.10. 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 minutes and a maximum of 10 minutes. The 3M Detection Chill Block Insert, used at ambient temperature without the Molecular Detection Chill Block Tray should sit directly on the laboratory bench. When cool, the lysis solution will revert to a pink color. 11.6.11. Remove the rack of LS tubes from the 3M Molecular Detection Chill Block Insert

11.7. Amplification 11.7.1. One Reagent tube is required for each sample and the NC. 11.7.1.1. Reagent tube strips can be cut to desired tube number. Select the number of individual Reagent tubes or 8-tube strips needed. 11.7.1.2. Place Reagent tubes in an empty rack. 11.7.1.3. Avoid disturbing the reagent pellets from the bottom of the tubes. 11.7.2. Select 1 Reagent Control (RC) tube and place in rack. 11.7.3. To avoid cross-contamination, decap one Reagent tube strip at a time and use a new pipette tip for each transfer step. 11.7.4. Transfer lysate to Reagent tubes and RC tube as described below: Transfer each sample lysate into individual Reagent tubes first followed by the NC. Hydrate the RC tube last. 11.7.5. Use the 3M Molecular Detection Cap/Decap Tool-Reagent to decap the Reagent tubes-one Reagent tube strip at a time. Discard cap. 11.7.5.1. Transfer 20 µL of Sample lysate from the upper ½ of the liquid (avoid precipitate) in the LS tube into corresponding Reagent tube. Dispense at an angle to avoid disturbing the pellets. Mix by gently pipetting up and down 5 times. 11.7.5.2. Repeat step 11.7.5.1 until individual sample lysate has been added to a corresponding Reagent tube in the strip. 11.7.5.3. Cover the Reagent tubes with the provided extra caps 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. 11.7.5.4. Repeat step 11.7.5.1 as needed, for the number of samples to be tested. 11.7.5.5. When all sample lysates have been transferred, repeat 11.7.4 to transfer 20 µL of NC lysate into a Reagent tube. 11.7.5.6. Transfer 20 µL of NC lysate into a RC tube. Dispense at an angle to avoid disturbing the pellets. Mix by gently pipetting up and down 5 times. 11.7.6. 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. 11.7.7. Review and confirm the configured run in the 3M Molecular Detection Software. 11.7.8. Click the Start button in the software and select instrument for use. The selected instrument’s lid automatically opens. 11.7.9. Place the 3M Molecular Detection Speed Loader Tray into the 3M Molecular Detection Instrument and close the lid to start the assay. Results are provided within 75 minutes, although positives may be detected sooner. 11.7.10. After the assay is complete, remove the 3M Detection Speed Loader Tray from the 3M Molecular Detection Instrument and dispose of the tubes by soaking in a 1-5% (v:v in water) household bleach solution for 1 hour and away from the assay preparation area.

Notice: To minimize the risk of false positives due to cross-contamination, never open reagent tubes containing amplified DNA. This includes Reagent Control, Reagent, and Matrix Control tubes. Always dispose of sealed reagent tubes by soaking in a 1-5% (v:v in water) household bleach solution for 1 hour and away from the assay preparation area.

11.8. Results and Interpretation An algorithm interprets the light output curve resulting from the detection of the nucleic acid amplification. Results are analyzed automatically by the software and are color-coded based on the result. A Positive or Negative result is determined by analysis of a number of unique curve parameters. Presumptive positive results are reported in real-time while Negative and Inspect results will be displayed after the run is completed.

Presumptive positive samples should be confirmed as per the laboratory standard operating procedures or by following the appropriate reference method confirmation, beginning with transfer from the primary enrichment to secondary enrichment broth (if applicable), followed by subsequent plating and confirmation of isolated using appropriate biochemical and serological methods.

NOTE: Even a negative sample will not give a zero reading as the system and 3M Molecular Detection Assay 2- E. coli O157:H7 amplification reagents have a “background” relative light unit (RLU).

In the rare event of any unusual light output, the algorithm labels this as “Inspect.” 3M recommends the user to repeat the assay for any Inspect samples. In the result continues to be Inspect, proceed to confirmation testing using FDA BAM.

1. CALCULATIONS Not applicable

2. QUALITY CONTROL 13.1. Reagent Purity: Accurate records are maintained, noting date received, opening dates, and shelf life for all reagents, solvents, and standards used in sample preparation and analysis. Any reagent that either contains interfering compounds or becomes contaminated with interfering compounds must be immediately discarded and replaced with an interference-free lot.

13.2. Evaluation of Negative control test: Each negative control test must demonstrate that the analytical system and reagents used in the analytical procedure are interference-free.

1. CALIBRATION AND STANDARDIZATION Not applicable

2. DATA ASSESSMENT AND ACCEPTANCE CRITERIA Data is continually assessed for completeness and accuracy.

3. CORRECTIVE ACTION FOR OUT-OF-CONTROL DATA The following are appropriate corrective actions to be taken in the event of out-of-control (OOC) data in the field. 16.1. Hold Time: Any sample analysis beyond the given hold time needs to be qualified and have it clearly stated in the comment section of the report. Samples that have been incubated for the proper time can be refrigerated for up to 4 days before running.

16.2. Proper temperature: If the incubator falls out of acceptable range 41.5±1°C then the incubator temperature must be adjusted by small increments until an acceptable temperature is reached. The incubator temperature must be recorded at each adjustment and after a half hour equilibration period. Samples which are influenced by failing temperature must be flagged.

1. SAFETY Safety glasses are required at all times in all laboratories. Protective gloves and lab coats are recommended in the laboratory for all sample preparation. On all projects, chemists need to be aware of and adhere to the New Age/Landmark Health and Safety Plan and any site-specific health and safety requirements.

Perform pathogen testing in a properly equipped laboratory under the control of trained personnel.

Always follow standard laboratory safety practices, including wearing appropriate protective apparel and eye protection while handling reagents and contaminated samples.

Avoid contact with the contents of the enrichment media and reagent tubes after amplification.

Always wear gloves (to protect the user and prevent introduction of nucleases).

1. POLLUTION PREVENTION AND WASTE MANAGEMENT Always dispose of the contaminated tubes by soaking in a 1-5% (v:v in water) household bleach solution for 1 hour and away from the assay preparation area.

Periodically decontaminate laboratory benches and equipment (pipettes, cap/decap tools, etc.) with a 1-5% (v:v in water) household bleach solution or DNA removal solution.

1. REFERENCES US Food and Drug Administration Bacteriological Analysis Manual. Chapter 4A: Diarrheagenic Escherichia coli. November 2015.

US Department of Agriculture (USDA) FSIS Microbiology Laboratory Guidebook 5.09. Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products and Carcass and Environmental Sponges. Effective Date: 15 January 2015.

ISO 16654:2001. Microbiology of Food and Animal Feeding Stuffs-Horizontal Method for the Detection of Escherichia coli O157.

ISO/IEC 17025. General requirements for the competence of testing and calibration laboratories.

ISO 7218. Microbiology of food and animal feeding stuffs-General rules for microbiological examination.

ISO 6887 Microbiology of food and animal feeding stuffs- Preparation of test samples, initial suspension and decimal dilutions for microbiological examination.

1. TABLES, DIAGRAMS, FLOWCHARTS, VALIDATION DATA

Table 2

EMPLOYEE RECEIPT AND ACKNOWLEDGMENT

I have received a copy of the above referenced New Age/Landmark, Inc. document. The document contains policies and rules, which apply to me. I have read the above referenced document, understand the contents of said document, and agree to comply with all documented procedures during my employment with New Age/Landmark, Inc. I further understand the document may be revised at any time, and that all changes will be communicated to me.