Validation of the Thermo Scientific SureTect^™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect^™ Escherichia coli STEC Identification PCR Assay for the Detection of Escherichia coli O157:H7 and the Escherichia coli STEC Serotypes (O26, O45, O103, O111, O121, O145) from Fresh Raw Spinach, Fresh Baby Leaves, Fresh Cut Tomatoes, Frozen Raw Beef, Raw Beef Trim, and Beef Carcass Sponges: AOAC Performance Tested Method^SM 012102

Abstract

Background

The Thermo Scientific SureTect^™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay are real-time PCR kits for the rapid detection of E. coli O157:H7 and non-E. coli O157 Shiga toxin-producing E. coli (STEC) serotypes (O26, O45, O103, O111, O121, O145) from fresh raw spinach, fresh baby leaves, fresh cut tomatoes, frozen raw beef, raw beef trim, and beef carcass sponges.

Objective

Both assays were evaluated for AOAC^®Performance Tested Methods^SM certification.

Methods

Detection and confirmation inclusivity/exclusivity, matrix, product consistency and stability, and robustness studies were conducted. In the matrix studies, the candidate method was validated against United States and international reference methods for STEC serotypes.

Results

Matrix studies showed no statistically significant differences between the candidate and reference method results when analyzed by probability of detection. For each inclusivity/exclusivity study, all inclusivity strains and no exclusivity strains were detected by either kit. Robustness testing demonstrated that the identification assay performed reliably despite method deviations; however, although not statistically significant, the screening assay performance was impacted. Product consistency and stability testing demonstrated no statistically significant differences between kit lots and storage time points.

Conclusion

The data presented show that both assays constitute a rapid and reliable workflow for the detection and confirmation of E. coli O157:H7 and stipulated non-E. coli O157:H7 STEC serotypes from the tested matrixes.

Highlights

Results are obtained in 80 min post-enrichment with both assays run simultaneously, allowing for the detection and confirmation of STEC within a single workflow.

General Information

Escherichia coli is a common, Gram-negative rod bacterium that is commonly part of the human gastrointestinal microbiome. Most E. coli are mutually symbiotic within the human intestinal tract; however, some strains can cause diarrhea or other organ system illnesses via opportunistic infection. Any strain of E. coli which produces a toxin known as Shiga toxin (stx) and an adhesin (eae) can cause diarrhea and are often transmitted through contaminated water and food and by contact with animals. These strains are collectively called Shiga toxin-producing E. coli (STEC) or enterohemorrhagic E. coli (EHEC). The Centers of Disease Control and Prevention (CDC) estimates that roughly one in six Americans (48 million people) become sick, 128 000 are hospitalized, and 3000 die from foodborne diseases each year. Symptoms of STEC vary from person to person, but often include severe stomach cramps, diarrhea, and vomiting. Some people may have a mild fever and some young children or elderly may develop hemolytic uremic syndrome (HUS) which requires hospitalization.

In the US, E. coli O157 is the most prevalent STEC serovar that contributes to STEC infections. There are a variety of foods that have been associated with E. coli O157:H7 outbreaks. Undercooked or raw hamburger (ground beef) has been implicated in many of the documented outbreaks, however E. coli O157:H7 outbreaks have also been linked to alfalfa sprouts, unpasteurized fruit juices, and other leafy produce. A total of six outbreaks have been linked to contaminated produce over the past five years. Other STEC serovars have also been implicated in outbreaks, with seven outbreaks linked to non-E. coli O157:H7 serovars. The non-O157:H7 serogroups O26, O45, O103, O111, O121, and O145 have been implicated as causative agents in multiple individual and outbreak cases and are therefore targets of interest (1).

Principle

The Thermo Scientific™ SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay are real-time PCR assays designed as a complete workflow for the rapid detection of Escherichia coli O157:H7 and other STEC serotypes (O26, O45, O103, O111, O121, O145). The assay is designed for use with the Applied Biosystems QuantStudio^™ 5 Real-Time PCR instrument and associated Applied Biosystems RapidFinder^™ Analysis software (version 1.1 or higher) (2).

The assay is supplied as a kit containing all necessary reagents to conduct the sample lysis, including prefilled lysis tubes and lyophilized PCR pellets, containing all necessary PCR reagents (target-specific primers, dye-labeled probes, and PCR master mix components) to easily conduct the PCR analysis of the sample. PCR probes are short oligonucleotides with a quencher molecule at one end that, when not bound to target DNA, greatly reduces fluorescence from the dye label at the opposite end of the probe molecule. Within the screening kit, the dye-labeled probes target unique DNA sequences specific to the E. coli STEC serotypes. If any E. coli STEC serotypes are present, the target DNA sequences will be amplified and the increasing fluorescent signal generated will be detected by the QuantStudio 5 Real-Time PCR instrument and interpreted by the RapidFinder Analysis software. The SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay contains four different channels (stx, eae, O157:H7 target 1, and O157:H7 target 2) that utilize different fluorophores to identify the presence of an STEC isolate. Both the stx and eae channels must have a positive result for the overall well call to be positive for the presence of an STEC. The O157:H7 target 1 and target 2 channels must be positive for an E. coli O157:H7 positive result, stx and eae channels may be positive or negative. The SureTect Escherichia coli STEC Identification PCR Assay should be run alongside the SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay. The identification kit differentiates between the non-E. coli O157:H7 STEC serotypes via unique fluorophores contained in each of the five channels, allowing for the presence of O26, O145, O103, O111, or O121/O45 (this is a single channel) to be characterized.

In addition to detection of any target DNA, the PCR pellets contain probes, primers, and DNA templates for an internal positive control (IPC). During PCR cycling, the IPC template is amplified regardless of whether target DNA is present or not. The probe used for the IPC is labeled with a different colored fluorescent dye to the probes used within the assay to detect target DNA, and so can be detected by the QuantStudio 5 Real-Time PCR instrument through a separate dye channel. If there is no presence of target DNA, the presence of the IPC amplification curve indicates that the PCR process has occurred successfully.

The PCR probes used in the SureTect Escherichia coli O157:H7 and STEC Screening and SureTect Escherichia coli STEC Identification PCR Kits are based on TaqMan^™ PCR technology. Results are achieved approximately 80 min after loading the prepared sample into the PCR instrument and are displayed via the RapidFinder Analysis software on the attached computer screen as simple positive or negative symbols with an attached PCR amplification plot that is easily accessible for review. All results interpreted by the software can be reported, stored, printed, and downloaded as required by the user.

Scope of Method

1. Analytes.—Shiga-toxin producing E. coli: E. coli O157:H7, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121, and E. coli O145.

2. Matrixes.—Fresh raw spinach (up to 375 g), fresh baby leaves (25 g), fresh cut tomatoes (25 g), frozen raw beef (25 g), raw beef trim (up to 375 g), and beef carcass sponge.

3. Summary of validated performance claims.—Comparable to the U. S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM), Chapter 4A (2018), Diarrheagenic Escherichia coli (3) for fresh raw spinach, the U. S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG), 5C.00, Detection, Isolation and Identification of Top Seven Shiga Toxin-Producing Escherichia coli (STECs) from Meat Products and Carcass and Environmental Sponges (4) for raw beef trim and beef carcass sponges, and the internal method from ADRIA Développement, based on ISO/TS 13136 (November 2012): Microbiology of food and animal feed—Real-Time Polymerase Chain Reaction (PCR)-based method for the detection of food-borne pathogens—Horizontal method for the detection of STEC and the determination of O157, O111, O26, O103, and O145 serogroups (5) for fresh baby leaves, fresh cut tomatoes, and raw ground beef. The Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay are valid confirmatory assays according to requirements in ISO 16140-6 Microbiology of the food chain—Method validation—Part 6: Protocol for the Validation of Alternative (Proprietary) Methods for Microbiological Confirmation and Typing Procedures (6).

Definitions

1. Probability of detection (POD).—The proportion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration. POD is concentration dependent. Several POD measures can be calculated: POD_R (reference method POD), POD_C (confirmed candidate method POD), POD_CP (candidate method presumptive result POD), and POD_CC (candidate method confirmation result POD).

2. Difference of probabilities of detection (dPOD).—Difference of probabilities of detection is the difference between any two POD values. If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

3. STEC.—Shiga-toxin producing E. coli

4. Stx 1.—Shiga-like toxin 1, a toxin produced by pathogenic E. coli that is closely related to Shiga toxin which is also produced by Shigella dysenteriae.

5. Stx 2.—Shiga-like toxin 2, a toxin produced by pathogenic E. coli that is closely related to Shiga toxin.

6. eae.—The gene associated with the attachment of E. coli to intestinal cells.

7. Intimin.—A virulence factor of EPEC and EHEC E. coli strains. This is an attaching and effacing protein coded for by the eae gene which causes enteropathogenic and enterohemorrhagic diarrhea by allowing the delivery of the Shiga toxin to cells.

Materials and Methods

Test Kit Information

1. Kit name.—Thermo Scientific™ SureTect^™ Escherichia coli O157:H7 and STEC Screening PCR Assay (Cat. No. A44254).

2. Kit Name.—Thermo Scientific™ SureTect^™ Escherichia coli STEC Identification PCR Assay (Cat. No. A45330).

3. Ordering information.

   • (1) USA.—Remel Inc., part of Thermo Fisher Scientific, Santa Fe Dr, Lenexa, KS, 66215. Tel: (800) 255-6730.

   • (2) Europe.—Oxoid Ltd, part of Thermo Fisher Scientific, Wade Rd, Basingstoke, Hampshire, RG24 8PW, UK. Tel: +44 1256 841144.

   • (3) Asia/Pacific/China.—Thermo Fisher Scientific, Thermo Fisher Biochemicals (Beijing) Ltd, 3^rd Floor, 28 Yuhua Rd, Area B, Tianzhu Airport Industrial Zone, Beijing 101312, China.

   • (4) Australia.—Thermo Fisher Scientific Australia Pty Ltd, 20 Dalgleish St, Thebarton, Adelaide, South Australia, 5031.

Test Kit Components

1. Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay.

2. Lysis Reagent 1 Tubes (clear, pale blue liquid containing fine white particles).—Twelve strips of eight tubes.

3. Lysis Tube Caps.—Twelve strips of eight domed caps.

4. Proteinase K (clear, colorless liquid).—One tube.

5. Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Tubes.—Twelve strips of eight tubes containing one lyophilized pellet each.

6. PCR Caps.—Twelve strips of eight caps.

7. SureTect Escherichia coli STEC Identification PCR Assay.—

   • (1) SureTect Escherichia coli STEC Identification PCR Tubes.—Twelve strips of eight tubes containing one lyophilized pellet each.

   • (2) PCR Caps.—Twelve strips of eight caps.

Additional Supplies and Reagents

Where sources are not referenced, items can be sourced from various suppliers.

1. Oxoid™ Buffered Peptone Water (ISO; BPW-ISO).—Cat. No. CM1211B or equivalent.

2. Oxoid Modified Tryptone Soya Broth (mTSB).—Cat. No. CM0989B.

3. Oxoid Sorbitol MacConkey Agar (CT-SMAC).—Cat. No. CMM813B or equivalent.

4. Oxoid C-T Supplement.—Cat. No. SR0172E or equivalent.

5. Escherichia coli O157 Latex Test.—Cat. No. DR0620M.

6. Remel™ RIM E. coli O157:H7 Latex Test.—Cat. No. R24250.

7. Dynabeads™ anti-E. coli O157, 1 mL.—Cat. No. 71003.

8. Dynabeads EPEC/VTEC O145, 2 mL.—Cat. No. 71007.

9. Dynabeads MAX EPEC/VTEC O45 Kit, 100 reactions.—Cat. No. A14631.

10. Dynabeads MAX EPEC/VTEC O121 Kit, 100 reactions.—Cat. No. A14632.

11. Dynabeads EPEC/VTEC O111, 2 mL.—Cat. No. 71009.

12. Dynabeads EPEC/VTEC O26, 2 mL.—Cat. No. 71013.

13. Dynabeads EPEC/VTEC O103, 2 mL.—Cat. No. 71011.

14. Commercially available latex agglutination kits for serogroups O26, O103, O111, O145.

15. Chromogenic Coliform Agar Dehydrated (CCA).—Cat. No. CM1205B.

16. CHROMagar™ STEC.—CHROMagar Cat. No. ST162.

Apparatus

Where sources are not referenced, the items are considered to be part of a microbiology laboratory as standard, these items can be sourced from various suppliers.

1. Homogenizer laboratory blender or dilutor.—DB5000A, DB 4100A, DB4150A, or equivalent. Available from Thermo Scientific Microbiology.

2. Homogenizer bags appropriate for the sample type and size.—For DB4100A or DB4150A: DB4011A, DB4012A, DB4013A, DB4014A, or equivalent.

3. Incubators fitted with racks for homogenizer bags, set to 41.5 ± 1°C.—For incubating enrichments.

4. Disposable gloves.—For handling samples.

5. Variable volume single-channel pipet, 1–10 mL.

6. 96-well rack.—For holding samples.

7. Filtered pipet tips, 1–10 mL.

8. Sample tubes, 1.5 mL.

9. Single-channel pipet or electronic, adjustable spacing, multichannel pipet, 10–100 µL.

10. Single-channel stepper pipet, 10–100 µL.

11. Filtered pipet tips, 10–100 µL.

12. Thermo Scientific CapEase™ tool for capping and decapping lysis tubes.—PT0621. Available from Thermo Scientific Microbiology.

13. Applied Biosystems SimpliAmp™ thermal cycler.—A24811. Available from Thermo Scientific Microbiology.

14. MicroAmp™ 96-Well Tray/Retainer Set for Veriti™ Systems.—4381850. Available from Thermo Scientific Microbiology.

15. MicroAmp Splash-Free 96-Well Base.—4312063. Available from Thermo Scientific Microbiology.

16. QuantStudio 5 Food Safety Real-Time PCR instrument, 0.1-mL block, with RapidFinder™ Analysis Software version 1.1 or later.—A36320 (desktop), A36328 (laptop). Available from Thermo Scientific Microbiology.

17. Vortex mixer.

18. 8-channel pipet, 10-100 µL.

19. Applied Biosystems QuantStudio 5 Spectral Calibration Plate (Cy^® 5.5 dye), 96-well 0.1 mL.—A45218. Available from Thermo Scientific Microbiology.

20. Plate centrifuge.

21. MicroAmp 96-Well Tray VeriFlex™ Block.—4379983. Available from Thermo Scientific Microbiology.

22. MicroAmp Splash-Free 96-Well Base.—4312063. Available from Thermo Scientific Microbiology.

23. Thermo Scientific 0.2-mL thin-walled 8-tube strips.—AB0452. Required to balance the lid pressure if less than two full strips are processed. Available from Thermo Scientific Microbiology.

24. Thermo Scientific Ultra Clear qPCR Caps, strips of 8.—AB0866. Required to balance the lid pressure if less than two full strips are processed. Available from Thermo Scientific Microbiology.

Reference Materials

Reference cultures were sourced from:

1. American Type Culture Collection (ATCC).—Manassas, VA, USA.

2. National Collection of Type Cultures (NCTC).—Salisbury, UK.

3. Michigan State University Culture Collection (MSU).—East Lansing, MI, USA.

4. Penn State University Culture Collection (PSU).—University Park, PA, USA.

5. Q Laboratories Culture Collection (QL).—Cincinnati, OH, USA.

6. Thermo Fisher Scientific, Microbiology Division, Research and Development Culture Collection (RDCC).—Basingstoke, UK.

7. Thermo Fisher Scientific, Microbiology Division, Trials Culture Collection (TCC).—Basingstoke, UK.

8. Thermo Fisher Scientific, Microbiology Division, Research and Development Culture Collection II (RDCCII).—Basingstoke, UK.

9. Thermo Fisher Scientific, Microbiology Division, Oxoid Culture Collection (OCC).—Basingstoke, UK.

10. Thermo Fisher Scientific Oy, Vantaa Culture Collection (VCC).—Vantaa, Finland.

11. Thermo Fisher Scientific, Lenexa Culture Collection (LEN).—Lenexa, KS, USA.

12. Agricultural Research Service Culture Collection (USDA).—Peoria, IL, USA.

13. ADRIA Développement (AD).—Quimper, France.

Safety Precautions

The following general precautions should always be followed. Clean workstations with disinfectant of choice (e.g., sodium hypochlorite solution, phenol solution, quaternary ammonium solution) before and after use as part of maintaining an aseptic technique. In addition to cleaning workstations, work areas should be separated for the following: media preparation, sample preparation, and pathogen detection. Gloves and other personal protective equipment should be used at all times, and the QuantStudio 5 instrument or supplies should never be touched without wearing gloves. When working with enrichments, filter laboratory bags should be used to minimize particulates and shaking the enrichment bag or collecting large food fragments should be avoided. For fatty foods, collect the sample just below the fat layer. Never reuse kit disposables and always change pipettes and pipet tips between samples. Product usage should follow good laboratory practices. All disposable materials must be discarded according to appropriate waste procedures used in the laboratory (2).

STEC are biosafety level 2 organisms and biological samples, such as enrichments, have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations on disposal of biological wastes. Wear appropriate protective equipment which includes but is not limited to protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities utilizing the appropriate safety equipment (for example, physical containment devices). Individuals should be trained in accordance with applicable regulatory and company/institution requirements before working with potentially infectious materials. All enrichment broths should be sterilized following any culture based confirmatory steps.

General Preparation

1. Prepare all media and use all confirmation kits according to the manufacturer’s instructions.

2. Turn on the QuantStudio instrument and the SimpliAmp thermal cycler and allow the systems to equilibrate. Ensure the SimpliAmp thermal cycler required temperature cycle is set according to the manufacturer’s instructions.

3. All lysis reagent and PCR tubes to reach room temperature before use by placing on the laboratory bench for approximately 10 min before they are required.

Sample Preparation

1. Fresh raw spinach (375 g).—For 375 g test portions, add 3375 ± 5 mL pre-warmed (41.5 ± 1°C) BPW-ISO. Homogenize by stomaching for 30 s to 1 min and incubate at 41.5 ± 1°C for 8–24 h.

2. Raw beef trim (375 g).—For 375 g test portions, add 1500 ± 5 mL pre-warmed (41.5 ± 1°C) mTSB. Homogenize by stomaching for 30 s to 1 min and incubate at 41.5 ± 1°C for 8–24 h.

3. Beef carcass sponges.—Wipe each surface area with a sampling sponge (World Bioproducts: EZ Reach Sponge Sampler with HiCap neutralizing buffer) 10 times vertically and 10 times horizontally. To each sponge add 200 mL pre-warmed (41.5 ± 1°C) mTSB. Homogenize by stomaching for 30 s to 1 min and incubate at 41.5 ± 1°C for 8–24 h.

4. Fresh cut tomatoes (25 g).—For 25 g test portions, add 225 mL (1-in-10 dilution) pre-warmed (41.5 ± 1°C) BPW, homogenize for 30 s to 1 min, and then incubate at 41.5 ± 1°C for 8–24 h.

5. Fresh baby leaves (25 g).—For 25 g test portions, add 225 mL (1-in-10 dilution) pre-warmed (41.5 ± 1°C) BPW, homogenize for 30 s to 1 min, and then incubate at 41.5 ± 1°C for 8–24 h.

6. Frozen raw beef (25 g).—For 25 g test portions, add 225 mL (1-in-10 dilution) of pre-warmed (41.5 ± 1°C) BPW, homogenize for 30 s to 1 min, and then incubate at 41.5 ± 1°C for 8–24 h.

Analysis

Lysis

1. Remove enriched sample from the incubator and mix thoroughly by shaking the bag and mixing thoroughly by hand for 30–60 s.

2. Remove the plastic seal from each Lysis Reagent 1 Tube, then add 10 µL of Proteinase K to the tube. Note: Avoid contamination of the Proteinase K stock tube. Use a new filtered pipet tip each time Proteinase K is withdrawn from the stock tube.

3. Transfer 10 µL enriched sample to a Lysis Tube. For the negative control, transfer 10 µL sterile enrichment media to a Lysis Tube. Ensure that the pipet tip reaches the bottom of the lysis tube, to facilitate complete mixing of the sample with Lysis Reagent 1.

4. Seal the tubes with domed Lysis Tube Caps. To conduct the lysis procedure, incubate the samples in the SimpliAmp thermal cycler set to the parameters outlined in Table  1. Note: To prevent crushing of the tubes in the Biosystems SimpliAmp Thermal Cycler, use the bottom piece from the MicroAmp 96-Well Tray/Retainer Set, or include at least four complete SureTect Lysis tube strips.

Table 1.

Biosystems SimpliAmp thermal cycler run parameters

Step	Temperature, °C	Time, min
Lid heater	105	Maximum time
1	37	10
2	95	5
3	10	2
4	4	Holda

^aFor convenience, samples can be held at 4°C until proceeding to PCR or transfer to storage at 2–8°C.

PCR analysis—SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay

1. Use the RapidFinder Analysis software to determine the run layout (plate layout) for samples based on the information entered and create a run file. Refer to the online help in the software for more details.

2. On the home page of the RapidFinder Analysis software, select “Create Experiment” then “enter” or “edit” the well parameters. Select “E. coli O157H7-STEC Screening SureTect QS5 version 1.1 or later” for the assay.

3. Following the plate layout previously set up in the software, place the required number of SureTect Escherichia coli O157:H7 and STEC Screening PCR Tubes (PCR tubes) in the MicroAmp 96-Well Tray for VeriFlex Block. Place the block on the MicroAmp Splash-Free 96-Well Base and press the PCR tubes to the tray to ensure they sit firmly, then tap the tubes on the bench to ensure that the pellets are located at the bottom of the tubes. If required by the plate layout, place empty tubes in the rack; these balance the tray when the tubes are placed in the instrument.

4. Allow the PCR tubes to remain on the bench for approximately 5 min to acclimatize to room temperature, then open one strip of PCR tubes by removing the seal. If the pellet is not positioned at the bottom of a tube, gently move the pellet to the bottom of the tube with a sterile, empty, pipet tip. Do not use a pipet tip containing lysate.

5. Uncap the Lysis Tubes using the CapEase decapping tool and transfer 20 µL lysate or mock-purified sample (negative extraction control reaction) to the appropriate PCR tube to rehydrate the pellet. Ensure that the lysate is removed from the top half of the liquid to ensure that no lysis particles are transferred from the Lysis Tube to the PCR tube as this can inhibit PCR from occurring. If particles become disturbed, leave the tube for 1–2 min to allow the particles to resettle. Do not touch the pellet when adding the lysate. Tap the rack gently to ensure that the lysate is at the bottom of the tube and touching the pellet.

6. Seal the PCR tubes with the flat optical PCR Caps provided with the kit, ensuring that the tubes are properly sealed by pressing down firmly over each opening, do not use the CapEase tool.

7. Mix all PCR tubes thoroughly for 10–15 s to ensure that the pellet is fully rehydrated. Ensure that the liquid is at the bottom of the tube before placing in the PCR instrument. If needed, hold the tubes upright, and flick sharply downward. The PCR run must be started within 30 min of the addition of sample lysates to the PCR tubes.

8. Eject the instrument drawer. Use the MicroAmp 96-Well Tray for VeriFlex Block to transfer the tubes to the instrument in the same configuration as the plate layout determined in the software, then close the instrument drawer.

9. In the “Run” tab of the experiment file in RapidFinder Analysis software, select the instrument’s serial number from the “Instrument” drop-down list.

10. Click “Start Run,” then follow the software prompts.

PCR analysis—SureTect Escherichia coli STEC Identification PCR Assay

1. Use the RapidFinder Analysis software to determine the run layout (plate layout) for samples based on the information entered and create a run file. Refer to the online help in the software for more details.

2. On the home page of the RapidFinder Analysis software, select “Create Experiment” then “enter” or “edit” the well parameters. Select “STEC Identification SureTect QS5 version 1.1 or later” for the assay.

3. Following the plate layout previously set up in the software, place the required number of SureTect Escherichia coli STEC Identification PCR Tubes (PCR tubes) in the MicroAmp 96-Well Tray for VeriFlex Block. Place the block on the MicroAmp Splash-Free 96-Well Base and press the PCR tubes to the tray to ensure they sit firmly, then tap the tubes on the bench to ensure that the pellets are located at the bottom of the tubes. If required by the plate layout, place empty tubes in the rack; these balance the tray when the tubes are placed in the instrument.

4. Allow the PCR tubes to remain on the bench for approximately 5 min to acclimatize to room temperature, then open one strip of PCR tubes by removing the seal. If the pellet is not positioned at the bottom of a tube, gently move the pellet to the bottom of the tube with a sterile, empty, pipet tip. Do not use a pipette tip containing lysate.

5. Uncap the Lysis Tubes using the CapEase decapping tool and transfer 20 µL of the lysate or mock-purified sample (negative extraction control reaction) to the appropriate PCR tube to rehydrate the pellet. Ensure that the lysate is removed from the top half of the liquid to ensure that no lysis particles are transferred from the Lysis Tube to the PCR tube as this can inhibit PCR from occurring. If particles become disturbed, leave the tube for 1–2 min to allow the particles to resettle. Do not touch the pellet when adding the lysate. Tap the rack gently to ensure that the lysate is at the bottom of the tube and touching the pellet.

6. Seal the PCR tubes with the flat optical PCR Caps provided with the kit, ensuring that the tubes are properly sealed by pressing down firmly over each opening, do not use the CapEase tool.

7. Mix all PCR tubes thoroughly for 10–15 s to ensure that the pellet is fully rehydrated. Ensure that the liquid is at the bottom of the tube before placing in the PCR instrument. If needed, hold the tubes upright and flick sharply downward. The PCR run must be started within 30 min of the addition of sample lysates to the PCR tubes.

8. Eject the instrument drawer. Use the MicroAmp 96-Well Tray for VeriFlex Block to transfer the tubes to the instrument in the same configuration as the plate layout determined in the software, then close the instrument drawer.

9. In the “Run” tab of the experiment file in RapidFinder Analysis software, select the instrument’s serial number from the “Instrument” drop-down list.

10. Click “Start Run,” then follow the software prompts.

Interpretation and test result report

When the run is complete, remove the processed samples from the QuantStudio 5 instrument and review the results on the computer screen. In the RapidFinder Analysis software, select “Results” on the main page to view results. Samples where PCR amplification takes place for the target analyte will be automatically interpreted by the software, and a positive result indicated by a red “+” symbol will be displayed on the plate layout. Where there has been no PCR amplification of the target analyte, a negative result indicated by a green “–” symbol will be displayed. In some cases with strong positive results, the software will return a positive call even when the IPC does not amplify. A yellow “!” symbol indicates that the sample has failed and should be repeated. See the RapidFinder Analysis software online help for options for reporting, export, and storage of results.

Confirmation

Samples with positive PCR results are presumptive and must be confirmed using the appropriate reference method for the sample matrix or by using the SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR alternative confirmation procedure.

1. Streak positive samples onto selective agar. For presumptive O157:H7 positives, streak 10 µL enrichment sample onto CT- SMAC agar, then incubate at 37 ± 1°C for 18–24 h. For presumptive non-O157:H7 positives, streak 10 µL enrichment sample onto either CCA or CHROMagar STEC agars, and then incubate at 37 ± 1°C for 18–24 h.

2. If presumptive colonies cannot be isolated, perform immunomagnetic separation according to the Dynabeads magnetic beads product instructions. Note: Ensure that the final elution volume of the concentrated sample is 100 µL.

3. Pipet, then streak 10 µL of the beads onto the agar and incubate as indicated previously. After isolating presumptive colonies on a selective agar, select a typical colony from the selective agar and emulsify in 1 mL diluent. If required, purify first a characteristic isolate onto a non-selective agar plate, e.g., tryptic soya agar (TSA). After preparation, perform lysis and PCR again using the SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay.

Validation Study

This validation study was conducted under the AOAC Research Institute (RI) Performance Tested Method(s)^SM program and the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces. Method developer studies were conducted in the laboratories of Thermo Fisher Scientific, Basingstoke, UK; Thermo Fisher Scientific Oy, Vantaa, Finland; and Thermo Fisher Scientific, Lenexa, KS, USA, and included product consistency and stability studies. Method developer studies also included a detection inclusivity/exclusivity study and a confirmation inclusivity/exclusivity study which was split with the independent laboratory, Q Laboratories (Cincinnati, OH, USA). Independent laboratory studies were conducted by Q Laboratories, Inc., USA and ADRIA Développement (Quimper, France). Q Laboratories conducted matrix studies for 375 g raw spinach, 375 g beef trim, and beef carcass sponges. Inclusivity/exclusivity studies for both detection and confirmation was split between Q Laboratories and the method developer laboratories. Robustness testing was conducted by Q Laboratories. ADRIA Développement conducted matrix studies for 25 g fresh cut tomatoes, 25 g fresh baby leaves, and 25 g frozen raw beef. The studies were conducted according to the AOAC guidelines for validation of microbiological methods following an unpaired study design.

Method Developer Studies

Detection inclusivity and exclusivity study

A total of 61 inclusivity isolates of E. coli STEC serotypes (containing stx1 and/or stx2 and eae) were tested. The 61 isolates comprised a minimum of eight strains per target serogroup. Of these 61 isolates, 35 were analyzed at Thermo Fisher Scientific, Basingstoke, UK, with the remaining 26 analyzed at Q Laboratories OH, USA, all using the QuantStudio 5 instrument. Isolates were obtained from internal culture collections with the origin of the strains representing a wide range of food, clinical, and environmental sources.

Inclusivity testing was conducted by removing isolates from –80°C storage and streaking onto non-selective agar for 18–24 h at 37°C before picking an isolate from the plate and then culturing each strain in BPW-ISO at 41.5 ± 1°C for 8 h. Each isolate was diluted in sterile enrichment media to approximately 100 times the LOD₅₀ (Level of detection for which 50% of tests gives a positive result) of the SureTect STEC procedure.

A total of 35 exclusivity isolates comprising of species closely related to E. coli and other E. coli non-target STEC strains were analyzed with the SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and the SureTect Escherichia coli O157:H7 and STEC Identification PCR Assay on the QuantStudio 5 instrument at Thermo Fisher Scientific, Basingstoke, UK. These isolates were obtained from internal culture collections. Exclusivity testing was conducted by removing isolates from −80°C storage, streaking onto a non-selective plate (TSA), and then incubating for 18–24 h at 37 ± 2°C. Each isolate was then inoculated into nutrient broth and cultured for 18–24 h at 37 ± 2°C.

Inclusivity and exclusivity isolates were blind-coded and randomized to ensure identity of the sample remained anonymous. The method showed 100% specificity, with all inclusivity and no exclusivity isolates detected, with the expected 13 O157:H7 isolates successfully excluded by the identification assay. The results for detection inclusivity and exclusivity are shown in Tables  2 and 3, respectively.

Table 2.

Detection inclusivity of the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and the SureTect Escherichia coli O157:H7 and STEC Identification PCR assay

Strain information						SureTect O157:H7 and STEC Screening Assay						SureTect STEC Identification PCR Assay
						stx	eae	O157 (target 1)	O157 (target 2)	IPC	Well call	O103	O111	O145	O26	O45/ O121	IPC	Well call
Location tested	Source	ID	Species	O group	Origin	Target call	Target call	Target call	Target call	Target call		Target call	Target call	Target call	Target call	Target call	Target call
Q Labs	MSU	TW07814	Escherichia coli	O26	Human	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	POSITIVE
Q Labs	MSU	TW04270	Escherichia coli	O26	Human	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	POSITIVE
Q Labs	MSU	TW07862	Escherichia coli	O26	Cow, calf	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	POSITIVE
Q Labs	MSU	TW04284	Escherichia coli	O26	Human child	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	POSITIVE
Q Labs	MSU	TW08101	Escherichia coli	O103	Human	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	POSITIVE
Q Labs	MSU	TW07971	Escherichia coli	O103	Human	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	POSITIVE
Q Labs	MSU	TW11239	Escherichia coli	O103	Human	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	POSITIVE
Q Labs	MSU	TW07697	Escherichia coli	O103	Human	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	POSITIVE
Q Labs	MSU	TW04162	Escherichia coli	O103	Human	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	POSITIVE
Q Labs	MSU	TW05614	Escherichia coli	O111	Human	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	POSITIVE
Q Labs	MSU	TW07502	Escherichia coli	O111	Human	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	POSITIVE
Q Labs	MSU	TW14960	Escherichia coli	O111	Human	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	POSITIVE
Q Labs	MSU	TW00186	Escherichia coli	O111	Human	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	POSITIVE
Q Labs	PSU	7.1711	Escherichia coli	O145	Not available	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	POSITIVE
Q Labs	PSU	10.0707	Escherichia coli	O145	Not available	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	POSITIVE
Q Labs	MSU	TW08039	Escherichia coli	O121	Human	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Q Labs	MSU	TW08023	Escherichia coli	O121	Human	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Q Labs	PSU	5.0959	Escherichia coli	O121	Not available	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Q Labs	PSU	7.1686	Escherichia coli	O121	Not available	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Q Labs	PSU	7.1709	Escherichia coli	O121	Not available	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Q Labs	MSU	TW09183	Escherichia coli	O45	Human	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Q Labs	MSU	TW10121	Escherichia coli	O45	Human	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Q Labs	MSU	TW01188	Escherichia coli	O45	Human	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Q Labs	MSU	TW01597	Escherichia coli	O45	Cow, calf	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Q Labs	PSU	6.3127	Escherichia coli	O45	Not available	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Q Labs	PSU	11.1079	Escherichia coli	O45	Not available	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Basa	RDCC	30033	Escherichia coli	O26	Unknown	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	POSITIVE
Bas	RDCC	30034	Escherichia coli	O26	Unknown	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	POSITIVE
Bas	RDCC	30204	Escherichia coli	O26	Bovine	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	POSITIVE
Bas	RDCC	30214	Escherichia coli	O26	Bovine	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	POSITIVE
Bas	RDCC	30037	Escherichia coli	O103	Unknown	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	POSITIVE
Bas	RDCC	30038	Escherichia coli	O103	Unknown	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	POSITIVE
Bas	RDCC	30164	Escherichia coli	O103	Unknown	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	POSITIVE
Bas	RDCC	30093	Escherichia coli	O111	Unknown	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	POSITIVE
Bas	RDCC	30200	Escherichia coli	O111	Unknown	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	POSITIVE
Bas	RDCC	30099	Escherichia coli	O111	Unknown	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	POSITIVE
Bas	RDCC	30173	Escherichia coli	O111	Unknown	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	POSITIVE
Bas	RDCC	30042	Escherichia coli	O145	Unknown	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	POSITIVE
Bas	RDCC	30043	Escherichia coli	O145	Unknown	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	POSITIVE
Bas	RDCC	30122	Escherichia coli	O145	Unknown	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	POSITIVE
Bas	RDCC	30123	Escherichia coli	O145	Unknown	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	POSITIVE
Bas	RDCC	30184	Escherichia coli	O145	Bovine	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	POSITIVE
Bas	RDCC	30185	Escherichia coli	O145	Unknown	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	POSITIVE
Bas	RDCC	30176	Escherichia coli	O121	Unknown	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Bas	RDCC	30178	Escherichia coli	O121	Unknown	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Bas	RDCC	30048	Escherichia coli	O121	Unknown	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Bas	RDCC	30210	Escherichia coli	O45	Unknown	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Bas	RDCC	30213	Escherichia coli	O45	Unknown	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	POSITIVE
Bas	RDCC	30071	Escherichia coli	O157:H7	Unknown	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVEb
Bas	RDCC	30072	Escherichia coli	O157:H7	Unknown	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30085	Escherichia coli	O157:H7	Unknown	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30088	Escherichia coli	O157:H7	Unknown	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30089	Escherichia coli	O157:H7	Unknown	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30090	Escherichia coli	O157:H7	Unknown	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30091	Escherichia coli	O157:H7	Unknown	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30156	Escherichia coli	O157:H7	Beef	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30193	Escherichia coli	O157:H7	Unknown	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30194	Escherichia coli	O157:H7	Unknown	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30196	Escherichia coli	O157:H7	Unknown	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30197	Escherichia coli	O157:H7	Unknown	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	913	Escherichia coli	O157:H7	Unknown	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE

^aBas = Tested at the method developer site, Thermo Fisher Scientific, Basingstoke, UK.

^bNEGATIVE = 13/13 expected negative results for O157:H7 isolates on the SureTect Identification assay.

Table 3.

Detection exclusivity of the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and the SureTect Escherichia coli O157:H7 and STEC Identification PCR assay

Strain information						SureTect O157:H7 and STEC Screening Assay						SureTect STEC Identification PCR Assay
						stx	eae	O157 (target 1)	O157 (target 2)	IPC		O103	O111	O145	O26	O45/ O121	IPC
Location tested	Source	ID	Genus/species	O group	Origin	Target call	Target call	Target call	Target call	Target call	Target call	Well call	Target call	Target call	Target call	Target call	Target call	Well call
Basa	RDCC	1167	Escherichia coli	O4	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	933	Escherichia coli	O128 VT	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	966	Escherichia coli	O91 VT	Unknown	+	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	2696	Escherichia coli	O113	Unknown	+	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	976	Escherichia coli	O1	Unknown	+	–	–	+	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1764	Escherichia coli	O48	Unknown	–	–	–	+	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1765	Escherichia coli	O62	Unknown	+	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1766	Escherichia coli	O74	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	2391	Escherichia coli	O163: H19 VT	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	644	Citrobacter freundii	N/Ab	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	963	Citrobacter freundii	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1208	Citrobacter freundii	N/A	Unknown	–	–	–	+	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	3715	Citrobacter braakii	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	255	Klebsiella aerogenes	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	648	Klebsiella aerogenes	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	557	Klebsiella pneumoniae	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	559	Klebsiella pneumoniae	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	953	Enterobacter cloacae	N/A	Unknown	+	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	954	Enterobacter cloacae	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	956	Enterobacter aerogenes	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	2966	Enterobacter aerogenes	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30077	Shigella dysenteriae	N/A	Unknown	–	+	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30078	Shigella dysenteriae	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30079	Shigella dysenteriae	N/A	Unknown	–	+	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	713	Shigella boydii	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	MHCC	5738	Shigella boydii	N/A	Unknown	–	+	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	1200	Salmonella arizonae	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	499	Shigella flexneri	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	1944	Shigella flexneri	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	MHCC	5584	Shigella flexneri	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	625	Shigella sonnei	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	1945	Shigella sonnei	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	2391	Shigella sonnei	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	722	Salmonella Typhimurium	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	723	Salmonella enteritidis	N/A	Unknown	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE

^aBas = Tested at the method developer site, Thermo Fisher Scientific, Basingstoke, UK.

^bN/A = Not applicable.

Confirmation inclusivity and exclusivity study

To validate the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay workflow as a confirmation method, an extensive inclusivity and exclusivity study following ISO 16140–6 was conducted for both assays. Work for this study was split between several method developer sites (Thermo Fisher Scientific, Basingstoke, UK; Lenexa, USA; and Vantaa, Finland) and with the independent laboratory, Q Laboratories, OH, USA. Several selective agars (CHROMagar STEC, CCA, and CT-SMAC) and TSA/blood agar were validated.

A total of 162 inclusivity isolates were analyzed with the SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay on the QuantStudio 5 instrument comprising of all STEC target serogroups—21 strains of O103, 25 strains of O111, 24 strains of O121, 17 strains of O145, 25 strains of O157:H7, 25 strains of O26, and 25 strains of O45.

Of these inclusivity isolates, 20 were tested at Thermo Fisher Scientific, Basingstoke, UK; 10 at Thermo Fisher Scientific Oy, Vantaa, Finland; 42 at Thermo Fisher Scientific, Lenexa, KS, USA; and the remaining 90 at Q Laboratories.

A total of 105 exclusivity strains were tested which comprised of 25 closely related non-E. coli strains and 85 non-target E. coli strains. The non-target E. coli strains consisted of a wide variety of O groups, including 10 strains of target O groups that lacked the stx1 and/or stx2 and eae gene.

Of these exclusivity isolates, 94 were tested at Thermo Fisher Scientific, Basingstoke, UK; one at Thermo Fisher Scientific, Lenexa, KS, USA; and 10 at Q OH, USA.

Isolates were obtained from national culture collections including ATCC and NCTC or sourced from other culture collections with the origin of the strains representing a wide range of food, clinical, and environmental sources.

Inclusivity isolates were prepared by removing isolates from –80°C storage and streaking onto each of the required agars—a non-selective, CCA, CHROMagar STEC, and CT-SMAC—and incubating for 37°C for 18–24 h.

Exclusivity isolates were prepared by streaking onto non-selective agar and incubating at 37°C for 18–24 h.

After incubation, an isolated colony was emulsified in 1 mL diluent and then prepared as a test sample by adding 10 µL to a lysis tube and mixing with Lysis Reagent 1 and Proteinase K. All samples were blind coded and randomized and then run through the SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay.

The SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay correctly detected all 162 inclusivity isolates, with the 25 O157:H7 isolates correctly excluded by the SureTect Escherichia coli STEC Identification PCR Assay. Several strains were not able to recover on CHROMagar STEC and/or CT-SMAC, but these isolates were picked up by the non-selective plate and CCA and correctly detected.

The SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay correctly excluded all 105 exclusivity isolates. The SureTect Escherichia coli STEC Identification PCR Assay correctly excluded the anticipated 100 strains and correctly identified the 10 non-STEC target O groups. The results of the confirmation inclusivity/exclusivity study are set out in Tables  4 and 5.

Table 4.

Confirmation inclusivity results for the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay results and SureTect Escherichia coli STEC Identification PCR assay

Strain information			Non-selective, CCA, CHROMagar STEC, and CT-SMAC resultsa
			SureTect O157:H7 and STEC Screening Assay						SureTect STEC Identification PCR Assay
			stx	eae	O157 (target 1)	O157 (target 2)	IPC	Well call	O103	O111	O145	O26	O45/ O121	IPC	Well call
Location	Isolate ID	O group	Target call	Target call	Target call	Target call	Target call		Target call	Target call	Target call	Target call	Target call	Target call
Basb	RDCC 30210	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Bas	RDCC 30213	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Lenc	LEN 341	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 195	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 331	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 332	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 338	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 425	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	USDA 222-5	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 426	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	USDA 261-1	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	USDA 622-1	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	USDA 4-C 95.3	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	USDA 4-C 96.3	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	USDA 600.1	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	USDA 606.3	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	USDA 634.1	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labsd	MSU TW09183	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	MSU TW10121	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	MSU TW14003	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	MSU TW07947	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	MSU DEC 11C	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	PSU 1.2635	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	PSU 6.3127	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	PSU 11.1079	O45	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Bas	RDCC 30033	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Bas	RDCC 30204	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Bas	RDCC 30214	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Bas	RDCC 30034	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Vane	VAN 210	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Van	VAN 165	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Len	LEN 225	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Len	LEN 216	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Len	LEN 217	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Len	LEN 218	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Len	LEN 219	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	ATCC BAA-1653	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU TW07814	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU TW00971	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU TW04270	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU TW04284	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU DEC9F	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU TW05992	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU TW08031	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU DEC9A	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU DEC 10B	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU DEC 10C	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU TW02295	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU TW07862	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	MSU DEC10E	O26	+	+	–	–	+	POSITIVE	–	–	–	+	–	+	O26
Q Labs	ATCC 35150	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	ATCC 43889	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	ATCC 43895	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	ATCC 51657	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	ATCC 51659	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	ATCC 700599	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	ATCC 700927	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU DEC3A	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU DEC3B	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU DEC3C	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU DEC3D	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU DEC3E	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU DEC4B	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU DEC4D	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU DEC4E	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU TW00116	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU TW00975	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU TW02302	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSUTW04863	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU TW05356	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	MSU TW07587	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	ATCC BAA-460	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	QL 2-202	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	QL 2-205	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	QL 2-206	O157:H7	+	+	+	+	+	POSITIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC 30184	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Bas	RDCC 30185	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Bas	RDCC 30122	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Bas	RDCC 30123	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Van	VAN 192	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Van	VAN 193	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Len	LEN 205	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Len	LEN 206	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Len	LEN 440f,g	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Q Labs	MSU TW09153	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Q Labs	MSU TW07596	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Q Labs	MSU TW01664	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Q Labs	MSU TW09356	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Q Labs	PSU 7.1711	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Q Labs	PSU 10.0707	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Q Labs	PSU 10.0708	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Bas	RDCC 30101	O145	+	+	–	–	+	POSITIVE	–	–	+	–	–	+	O145
Bas	RDCC 30048	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Van	VAN 214	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 201	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 202	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 203	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 328f	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 358	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 359	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	LEN 360	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	USDA 63-1/429	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	USDA 1-C 10.2/430	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	USDA 1-C 16.3/431	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Len	USDA 1-C 21.2/432	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	MSU TW07614	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	MSU TW08023	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	MSU TW08039	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	MSU TW07931	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	MSU MT#2	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	PSU 5.0959	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	PSU 7.1686	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	PSU 7.1709	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	PSU 7.1732	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Q Labs	PSU 10.0709	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Bas	RDCC 30176	O121	+	+	–	–	+	POSITIVE	–	–	–	–	+	+	O45/O121
Bas	RDCC 30173	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Bas	RDCC 30093	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Bas	RDCC 30200	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Bas	RDCC 30099	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Van	VAN 195	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Van	VAN 196	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Len	LEN 200	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Len	LEN 252	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Len	LEN 327f	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Len	LEN 334	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Len	LEN 198f	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU TW07926	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU DEC8D	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU TW14960	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU TW06296	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU TW06315	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU DEC12A	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU DEC15A	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU TW05614	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU TW00186	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU TW01387	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU TW07502	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU DEC6A	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	MSU DEC6C	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Q Labs	QL 12289-3A	O111	+	+	–	–	+	POSITIVE	–	+	–	–	–	+	O111
Bas	RDCC 30037	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Bas	RDCC 30038	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Van	VAN 134	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Van	VAN 190	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Van	VAN 212	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Len	LEN 270	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Len	LEN 196	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Len	LEN 197f,g	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Q Labs	MSU TW09101	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Q Labs	MSU TW07971	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Q Labs	MSU TW11239	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Q Labs	MSU TW07697	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Q Labs	MSU TW05997	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Q Labs	MSU TW04162	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Q Labs	MSU TW08101	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Q Labs	NCTC 8196	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Q Labs	PSU 5.1658	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Q Labs	PSU 7.1691	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Q Labs	PSU 9.0036	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Q Labs	QL15071-2	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103
Bas	RDCC 30164	O103	+	+	–	–	+	POSITIVE	+	–	–	–	–	+	O103

^aResults were equivalent for all agars tested unless otherwise indicated by “^f” or “^g”.

^bBas = Tested at the method developer site, Thermo Fisher Scientific, Basingstoke, UK.

^cLen = Tested at method developer site, Thermo Fisher Scientific, Lenexa, KS, USA.

^dQ Labs = Tested at Independent Laboratory, Q Laboratories Inc., Cincinnati, OH, USA.

^eVan = Tested at method developer site, Thermo Fisher Scientific Oy, Vantaa, Finland.

^fIsolate failed to recover on CHROMagar STEC media.

^gIsolate failed to recover on CT-SMAC media.

Table 5.

Confirmation exclusivity results for the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay results and SureTect Escherichia coli STEC Identification PCR assay—non-selective agar

Strain information					Non-selective agar
					SureTect O157:H7 and STEC Screening Assay						SureTect STEC Identification PCR Assay
					stx	eae	O157 (target 1)	O157 (target 2)	IPC	Well call	O103	O111	O145	O26	O45/ O121	IPC	Well call
Location	Culture collection	Strain ID	Organism	O group	Target call	Target call	Target call	Target call	Target call		Target call	Target call	Target call	Target call	Target call	Target call
Basa	RDCC	1167	E. coli HG2	O4	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	933	E. coli HG3	O128 VT-	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	966	E. coli HG4	O91 VT-	+	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	2696	E. coli HG5	O113	+	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	976	E. coli HG6	O1	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1764	E. coli HG7	O48	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1765	E. coli HG8	O62	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1766	E. coli HG9	O74	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	2391	E. coli HG10	O163: H19 VT-	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	644	C. freundii	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	963	C. freundii	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1208	C. freundii	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	255	K. aeorgenes	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	648	K. aeorgenes	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	557	K. pneumoniae	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	559	K. pneumoniae	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	953	E. cloaecae	N/A	+	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	954	E. cloaecae	N/A	–	–	–	+	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	956	E. aerogenes	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	2966	E. aerogenes	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30077	S. dysenteriae	N/A	–	+	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30078	S. dysenteriae	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	30079	S. dysenteriae	N/A	–	+	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	713	S. boydii	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCCII	5738	S. boydii	N/A	–	+	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	2700	S. boydii	N/A	–	–	–	+	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	499	S. flexneri	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	1944	S. flexneri	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCCII	5584	S. flexneri	N/A	–	–	–	+	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	625	S. sonnei	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	1945	S. sonnei	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	OCC	2391	S. sonnei	N/A	–	–	–	+	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	962	S. Typhimurium	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	961	S. enteritidis	N/A	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1259	E. coli	O116	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1769	E. coli	O125ab	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1796	E. coli	O116	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	4731	E. coli	E O103: K: K8	–	–	–	–	+	NEGATIVE	+	–	–	–	–	+	O103
Bas	RDCC	4732	E. coli	O76	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	4733	E. coli	O31	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	4734	E. coli	O129	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	4735	E. coli	O19a	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	848	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	973	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	974	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	975	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	993	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1122	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1723	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1744	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1745	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1746	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1747	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1748	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1776	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	1995	E. coli	O111 (NCTC 9703)	–	+	–	–	+	NEGATIVE	–	+	–	–	–	+	O111
Bas	RDCC	3468	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	3520	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	3717	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	4254	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	4257	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	4675	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	4723	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	4725	E. coli		–	+	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	4726	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	4727	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	4729	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	5939	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	5941	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	5943	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	5944	E. coli		–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	909	E. coli	O91: H21 VT2	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	934	E. coli	O128 VT neg	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	935	E. coli	O128 VT neg	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	936	E. coli	O128 VT neg	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	937	E. coli	O128 VT neg	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	938	E. coli	O128 VT neg	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	967	E. coli	O91 VT neg	+	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	2695	E. coli	O91: H21	+	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	944	E. coli	O128 VT neg	–	+	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	945	E. coli	O128 VT neg	–	+	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	968	E. coli	O91 VT neg	+	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	951	E. coli	O128 VT neg	–	+	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	952	E. coli	O128 VT neg	–	+	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	953	E. coli	O128 VT neg	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	TCC	954	E. coli	O128 VT neg	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	2399	E. coli	O128	–	+	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Bas	RDCC	5791	E. coli	O45	–	–	–	–	+	NEGATIVE	–	–	–	–	+	+	O45/O121
Bas	RDCC	5792	E. coli	O121	–	–	–	–	+	NEGATIVE	–	–	–	–	+	+	O45/O121
Bas	RDCC	5795	E. coli	O103: K+:H8	–	–	–	–	+	NEGATIVE	+	–	–	–	–	+	O103
Bas	RDCC	5796	E. coli	O111: H2	–	+	–	–	+	NEGATIVE	–	+	–	–	–	+	O111
Bas	RDCC	5797	E. coli	O26: K60: H-	–	+	–	–	+	NEGATIVE	–	–	–	+	–	+	O26
Bas	RDCC	30178	E. coli	O121	–	+	–	–	+	NEGATIVE	–	–	–	–	+	+	O45/O121
Bas	OCC	199	E. coli	O6	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Lenb	LEN	395	E. coli	O121	+	–	–	–	+	NEGATIVE	–	–	–	–	+	+	O45/O121
Q Labsc	Q Labs	MSU TW00585	E. coli	O55	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	Q Labs	MSU DEC1A	E. coli	O55: H6	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	Q Labs	NCTC 9091	E. coli	O91	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	Q Labs	NCTC 9113	E. coli	O113	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	Q Labs	NCTC 10444	E. coli	O115	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	Q Labs	NCTC 9117	E. coli	O117	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	Q Labs	NCTC 9118	E. coli	O118	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	Q Labs	NCTC 10089	E. coli	O142	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	Q Labs	MSU 10677	E. coli	O146	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE
Q Labs	Q Labs	11021	E. coli	O163	–	–	–	–	+	NEGATIVE	–	–	–	–	–	+	NEGATIVE

^aBas = Tested at the method developer site—Thermo Fisher Scientific, Basingstoke, UK.

^bLen = Tested at method developer site—Thermo Fisher Scientific, Lenexa, KS, USA.

^cQ Labs = Tested at independent laboratory—Q Laboratories Inc., Cincinnati, OH, USA.

Product consistency (lot-to-lot) and stability studies

The product consistency and stability study examined three lots of the SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and the SureTect Escherichia coli O157:H7 and STEC Identification PCR Assay kits for lot-to-lot variability and product stability to confirm that the manufacturing and performance of the kits were consistent at different stages during shelf life. The three kits consisted of one lot near the expiration date (END1), one near the middle of the expiration period (MID), and one lot that had been recently manufactured (FRESH).

One strain of E. coli O157:H7 (used to assess the SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay) and one strain of E. coli O26 (used to assess the SureTect Escherichia coli O157:H7 and STEC Identification PCR Assay) were cultured in BPW-ISO at 37.0°C for 8 h. This was then diluted in sterile enrichment buffer to a concentration at the limit of detection (LOD₅₀) of the SureTect STEC assays to achieve fractional recovery.

Citrobacter freundii was used as a closely related non-target strain and was cultured in non-selective broth at 37°C for 24 h and not diluted.

Each kit lot was tested with 10 replicates each of E. coli O157:H7, and 10 replicates of the undiluted C. freundii in a randomized blind-coded fashion.

POD values and confidence intervals were calculated from the data to determine any statistical variation between lots. All C. freundii samples were negative for all replicates for all three kits.

Fluorescence levels were comparable between kit lots and there was no significant difference between the kits at the 5% confidence level. The POD analysis of the stability study is outlined in Tables  6 and 7.

Table 6.

Product consistency (lot-to-lot) and stability of Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay method—POD comparison

Kit Lot No.	Lot age	n a	x b	${\text{POD}}_{\mathrm{A}}$ c	95% CI	Kit Lot No.	Lot age	n	x	${\text{POD}}_{\mathrm{B}}$ d	95% CI	${\text{dPOD}}_{\text{AB}}$ e	95% CIf
E. coli O26 (target)
0020001500	Freshg	10	7	0.7	0.40, 0.89	0020000860	Middle	10	5	0.5	0.24, 0.76	–0.20	–0.53, 0.20
0020000860	Middleh	10	5	0.5	0.24, 0.76	Prelaunch A	End	10	5	0.5	0.24, 0.76	0.00	–0.37, 037
Prelaunch A	Endi	10	5	0.5	0.24, 0.76	0020001500	Fresh	10	7	0.7	0.40, 0.89	0.20	–0.20, 0.53
Citrobacter freundii (non-target)
0020001500	Fresh	10	0	0.00	0.00, 0.28	0020000860	Middle	10	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
0020000860	Middle	10	0	0.00	0.00, 0.28	Prelaunch A	End	10	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
Prelaunch A	End	10	0	0.00	0.00, 0.28	0020001500	Fresh	10	0	0.00	0.00, 0.28	0.00	–0.28, 0.28

^a n = Number of test portions.

^b x = Number of positive test portions.

^cPOD_A = Positive outcomes divided by the total number of trials first member of pair.

^dPOD_B = Positive outcomes divided by the total number of trials second member of pair.

^edPOD_AB = Difference in POD between the paired comparison.

^f95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

^gFresh = 2 months old.

^hMiddle = 7 months old.

ⁱEnd = 10 months old.

Table 7.

Product consistency (lot-to-lot) and stability of SureTect Escherichia coli STEC Identification PCR Assay method—POD comparison

Kit Lot No.	Lot age	n a	x b	${\text{POD}}_{\mathrm{A}}$ c	95% CI	Kit Lot No.	Lot age	n	x	${\text{POD}}_{\mathrm{B}}$ d	95% CI	${\text{dPOD}}_{\text{AB}}$ e	95% CIf
E. coli O26 (target)
0020001482	Freshg	10	8	0.80	0.49, 0.94	0020000862	Middle	10	7	0.70	0.40, 0.89	–0.10	–0.44, 0.26
0020000862	Middleh	10	7	0.70	0.40, 0.89	Prelaunch B	End	10	9	0.90	0.60, 1.00	0.20	–0.16, 0.52
Prelaunch B	EndI	10	9	0.90	0.60, 1.00	0020001482	Fresh	10	8	0.80	0.49, 0.94	–0.10	–0.43, 0.24
Citrobacter freundii (non-target)
0020001482	Fresh	10	0	0.00	0.00, 0.28	0020000862	Middle	10	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
0020000862	Middle	10	0	0.00	0.00, 0.28	Prelaunch B	End	10	1	0.10	0.00, 0.40	0.10	–0.20, 0.40
Prelaunch B	End	10	1	0.10	0.00, 0.40	0020001482	Fresh	10	0	0.00	0.00, 0.28	–0.10	–0.20, 0.40

^a n = Number of test portions.

^b x = Number of positive test portions.

^cPOD_A = Positive outcomes divided by the total number of trials first member of pair.

^dPOD_B = Positive outcomes divided by the total number of trials second member of pair.

^edPOD_AB = Difference in POD between the paired comparison.

^f95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

^gFresh = 2 months old.

^hMiddle = 7 months old.

^IEnd = 10 months old.

Independent Laboratory Studies

The independent Laboratory studies were conducted by Q Laboratories, USA and ADRIA. Q Laboratories conducted detection inclusivity/exclusivity testing; confirmation inclusivity/exclusivity testing; matrix studies for 375 g fresh raw spinach, 375 g beef trim, and beef carcass sponges; and robustness testing. ADRIA conducted matrix studies for 25 g fresh cut tomatoes, 25 g baby leaves, and 25 g frozen raw beef.

Detection inclusivity/exclusivity studies

A total of 61 inclusivity isolates of E. coli STEC serotypes (containing stx1 and/or stx2 and eae) were tested comprising a minimum of eight strains per serogroup. Of these 61 isolates, 26 were analyzed at Q Laboratories Inc. using the QuantStudio 5 instrument. Isolates were obtained from internal culture collections with the origin of the strains representing a wide range of food, clinical, and environmental sources.

Inclusivity testing was conducted by removing isolates from –80°C storage and culturing each strain on a non-selective plate before isolates were subbed from the plate and cultured in BPW-ISO at 41.5 ± 1°C for 8 h. Each isolate was diluted in sterile enrichment media to approximately 100 times the LOD₅₀ of the SureTect STEC procedure.

All 35 exclusivity isolates were analyzed at the method develop laboratory (Thermo Fisher Scientific, Basingstoke, UK).

The method showed 100% specificity, correctly identifying all inclusivity isolates tested at Q Laboratories. The results for detection inclusivity and exclusivity are shown in Tables  2 and 3, respectively.

Confirmation inclusivity/exclusivity studies

The Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay workflow underwent an extensive inclusivity and exclusivity study following ISO 16140-6 to validate the workflow as a confirmation method. Work for this study was split between several method developer sites (Basingstoke, UK; Lenexa, KS, USA; and Vantaa, Finland) and also with the independent laboratory, Q Laboratories. Several selective agars (CHROMagar STEC, CCA, and CT-SMAC) and one non-selective agar were validated.

A total of 162 inclusivity isolates were analyzed with the SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay on the QuantStudio 5 instrument comprising of all STEC target serogroups—21 strains of O103, 25 strains of O111, 24 strains of O121, 17 strains of O145, 25 strains of O157:H7, 25 strains of O26, and 25 strains of O45. Of the 162 inclusivity isolates, 90 were tested at Q Laboratories.

A total of 105 exclusivity strains were tested which comprised of 25 closely related non-E. coli strains and 85 non-target E. coli strains. The non-target E. coli strains consisted of a wide variety of O groups, including 10 strains of target O groups that lacked the stx1/stx2 and eae gene. Of the 105 exclusivity isolates, 10 were tested at Q Laboratories.

Isolates were obtained from the NCTC, MSU, and QL. Inclusivity isolates were prepared by streaking onto each of the required agars—a non-selective, CCA, CHROMagar STEC, and CT-SMAC—and incubating for 37°C for 18–24 h. Exclusivity isolates were prepared by streaking onto non-selective agar and incubating at 37°C for 18–24 h. After incubation, an isolated colony was emulsified in 1 mL diluent and then prepared as a test sample by adding 10 µL to a lysis tube and mixing with Lysis Reagent 1 and Proteinase K. All samples were blind coded and randomized and then run through the SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay.

The SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay correctly detected all 90 inclusivity isolates tested, with the 25 O157:H7 isolates correctly excluded by the SureTect Escherichia coli STEC Identification PCR Assay. The SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay successfully excluded all 10 exclusivity isolates. The results of the confirmation inclusivity/exclusivity study are set out in Tables  4 and 5.

Matrix study—Q Laboratories

The matrix study was conducted according to the AOAC guidelines for validation of microbiological methods following the AOAC RI Performance Tested Methods^SM following an unpaired study design for 375 g fresh raw spinach, 375 g beef trim, and beef carcass sponge.

For the matrix study, the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay was compared to the FDA/BAM Chapter 4A reference method for 375 g fresh raw spinach, and the USDA/FISIS-MLG 5C.00 reference method for 375 g raw beef trim and beef carcass sponges.

Fresh raw spinach was purchased from a local supplier and prescreened for natural contamination of STEC organisms following the FDA/BAM Chapter 4A reference method. Raw beef trim and raw beef brisket (used for beef carcass sponges) were purchased from a local supplier and prescreened for natural contamination of STEC organisms following the USDA/FISIS-MLG 5C.00 reference method. Total aerobic plate count was determined following the FDA/BAM Chapter 3 Aerobic Plate Count reference method for fresh raw spinach (7) and following the USDA/FSIS-MLG 3.02 Quantitative Analysis of Bacteria in Foods as Sanitary Indicators reference method for raw beef trim and raw beef brisket (used for beef carcass sponges; 8). No natural contamination was detected in the screening of all three products therefore matrixes were artificially contaminated as follows: E. coli O157:H7 ATCC 51657 was used to inoculate 375 g fresh raw spinach, E. coli O45 MSU DEC11C was used to inoculate 375 g raw beef trim, and E. coli O111 MSU TW07926 was used to inoculate raw beef brisket (used for beef carcass sponges).

The matrix study conducted at Q Laboratories consisted of evaluating a total of 30 unpaired 375 g samples and 30 unpaired 25 g samples for fresh raw spinach and fresh raw beef trim. Within each sample set, there were 5 uninoculated samples (0 CFU/test portion), 20 low-level inoculated samples (0.2–2 CFU/test portion), and 5 high-level inoculated samples (2–10 CFU/test portion). A total of 30 unpaired sponges were also evaluated for the beef carcass sponges. Within each sample set, there were 5 uninoculated samples (0 CFU/sponge), 20 low-level inoculated samples (0.2–2 CFU/sponge), and 5 high-level inoculated samples (2–10 CFU/sponge).

All isolates used to inoculate the 375 g fresh raw spinach and 375 g raw beef trim were prepared by propagating from a stock culture stored at –70°C to TSA with 5% sheep blood agar (SBA) and incubating for 24 ± 2 h at 35 ± 1°C. A single colony was then transferred into brain heart infusion (BHI) broth and incubated at 35 ± 1°C for 24 ± 2 h. Following incubation, the culture was diluted to a target level using BHI as the diluent and added to the matrix at an appropriate amount where the low-level (0.2–2 CFU/test portion) inoculated samples would yield expected fractional positive results (5–15 positive results) and the high-level (2–10 CFU/test portion) inoculated samples would yield expected all-positive results. Inoculated matrix was mixed to ensure homogeneous distribution of the organisms within the matrix and was held for 48–72 h at 2–8°C to allow for equilibration of the organism as per AOAC guidelines. For the 375 g test portions, 25 g from each contamination level was combined with 350 g of uninoculated matrix on the day of analysis. For 200 g test portions, 25 g from each contamination level was combined with 175 g of uninoculated matrix on the day of analysis. For 25 g test portions, 25 g replicates for each level of contamination were transferred to sterile filter laboratory blender bags on the day of analysis.

For the beef carcass sponges, inoculum was prepared by propagating from a stock culture stored at –70°C to SBA and incubating for 24 ± 2 h at 35 ± 1°C. A single colony was then transferred into BHI broth and incubated at 35 ± 1°C for 24 ± 2 h. Following incubation, the culture was diluted to a target level using BHI as the diluent and spread evenly with an L-shaped spreader across a 4 × 4” surface area of raw beef brisket at an appropriate amount where the low-level (0.2–2 CFU/sponge) inoculated samples would yield expected fractional positive results (5–15 positive results) and the high-level (2–10 CFU/sponge) inoculated samples would yield expected all-positive results. The matrix was held for 48–72 h at 2–8°C prior to sampling to allow for equilibration of the organism as per current AOAC guidelines.

All test portions were prepared and incubated according to the protocol described in the Sample Preparation section. All samples were analyzed by following the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay at 8 and 24 h time points for the 375 g fresh raw spinach, 375 g raw beef trim and beef carcass sponges. All samples were analyzed using the QuantStudio 5 instrument and results were interpreted with the RapidFinder Analysis software. Following the 24 h time point, all samples regardless of presumptive results were culturally confirmed by the FDA/BAM Chapter 4A reference method for 375 g fresh raw spinach, and the USDA/FISIS-MLG 5C.00 reference method for raw beef trim and beef carcass sponges. Confirmation at the E. coli species level was achieved using Official Methods of Analysis^SM Method 2011.17 (9). In addition, the alternative SureTect STEC confirmation procedure of the genotypes was performed as previously described in the Confirmation section.

FDA/BAM Chapter 4A reference method.—For the FDA/BAM Chapter 4A reference method, 200 g test portions of fresh raw spinach were enriched with 450 ± 5 mL modified buffered peptone water with pyruvate and acriflavin, cefsulodin, and Vancomycin supplement (mBPWp + ACV). All test portions were not stomached or blended. All samples were incubated for 18–24 h at 42 ± 1°C.

Following enrichment, all test portions were serially diluted 1 in 10 in phosphate-buffered water (PBW) to 10⁻² through 10⁻⁴. A 100 µL aliquot of the serial dilutions were plated in duplicate onto CT-SMAC and CCA in order to achieve isolated colonies. All plates were incubated for 18–24 h at 37 ± 1°C. After incubation, plates containing typical colonies were screened for the appropriate STEC by latex agglutination.

Up to 10 isolated colonies that screened positive were streaked to SBA and TSA with yeast (TSA/YE) and incubated for 18–24 h at 37 ± 1°C. Following incubation, a ColiComplete (CC) disc was placed into the heaviest area of growth on the TSA/YE plates and incubated for an additional 18–24 h at 37 ± 1°C. The CC discs on the TSA/YE plates were observed for typical reactions (blue color change with no fluorescence under long wave UV) and a spot indole test was conducted. Biochemical confirmations of the E. coli species were obtained by VITEK 2 GN biochemical identification following AOAC OMA 2011.17.

USDA/FSIS-MLG 5C.00 reference method.—The USDS/FSIS-MLG 5C.00 reference method was modified by only performing the final identification of the O group for positive samples using the iQ-Check™ SerO from mRBA plates only, instead of performing identification from both mRBA plates and SBA plates. For the USDA/FISIS-MLG 5C.00 reference method, the 25 g test portions were analyzed as unpaired test portions with the alternative method. The 25 g test portions of fresh raw beef trim were enriched with 225 ± 4.5 mL mTSB. Samples were massaged by hand for 20–30 s to disperse clumps and incubated for 15–24 h at 42 ± 1°C. After incubation, all samples were screened for stx and eae genes using the iQ-Check VirX following the user guide and product instructions. Samples that screened negative for the stx and/or eae gene were reported as negative, inconclusive results were repeated, and all samples that screened positive for stx and eae genes were confirmed by immunomagnetic separation (IMS) isolation with the appropriate anti-E. coli magnetic beads and plated onto mRBA. mRBA plates were incubated at 35 ± 2° C for 20–24 h. Following incubation, mRBA plates were examined for colonies and latex agglutination was performed to confirm typical colonies. Samples that had no growth or were agglutination-negative were reported as negative. Samples that agglutinated and tested positive for appropriate O groups were streaked to SBA for isolation. SBA plates were incubated at 35 ± 2°C for 20–24 h. Colonies that were agglutination-positive were further confirmed for serogroup by using the iQ-Check SerO. To confirm using the iQ-Check SerO, the remainder of the agglutinating colony was transferred and homogenized in 50 µL molecular-grade water. A 5 µL aliquot of the suspension was added to 20 µL iQ-Check SerO PCR mix. The iQ-Check SerO was run following the user guide and product instructions.

Following incubation, latex agglutination was performed. Confirmation at the species level for all samples was achieved by AOAC Method 2011.17.

The level of E. coli O157:H7 in the low-level inoculum for all 200 g test portions of fresh raw spinach was determined by most probable number (MPN) on the day of analysis by evaluating 5 × 400 g, 20 × 200 g (reference method test portions), and 5 × 100 g inoculated test samples. The level of E. coli O157:H7 in the high-level inoculum for all 200 g test portions of fresh raw spinach was determined by MPN by evaluating 5 × 200 g (reference method test portions), 5 × 100 g, and 5 × 50 g inoculated test samples. To the 400 g portions, 900 mL reference method enrichment broth was added; to the 100 g portions, 225 mL reference method enrichment broth was added; and to the 50 g portions, 112.5 mL enrichment broth was added.

The level of E. coli O45 in the low-level inoculum for all 25 g test portions of raw beef trim was determined by MPN on the day of analysis by evaluating 5 × 50 g, 20 × 25 g (reference method test portions), and 5 × 10 g inoculated test samples. The level of E. coli O45 in the high-level inoculum for all 225 g test portions of raw beef trim was determined by MPN by evaluating 5 × 25 g (reference method test portions), 5 × 10 g, and 5 × 5 g inoculated test samples. To the 50 g portions, 450 mL reference method enrichment broth was added; to the 10 g portions, 90 mL reference method enrichment broth was added; and to the 5 g portions, 45 mL enrichment broth was added.

All 200 g portions of fresh raw spinach were utilized from unpaired reference method test potions analyzed following the FDA/BAM Chapter 4A reference method. All 25 g portions of raw beef trim were utilized form unpaired reference method test portions analyzed following the USDA/FISIS-MLG 5C.00. The number of positives from the three test levels was used to calculate the MPN using the LCF MPN calculator (version 1.6) provided by AOAC RI (10).

As per criteria outlined in Appendix J of the Official Methods of Analysis Manual, fractional positive results were obtained for fresh raw spinach (375 g test portions) at 8 and 24 h time points, and 8 and 24 h time points for raw beef trim (375 g test portions) and beef carcass sponges for the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and the SureTect Escherichia coli STEC Identification PCR Assay (11). Prior to inoculation, an aerobic plate count (APC) result of 3.2 × 10⁴ CFU/g was obtained from the fresh raw spinach, 4.2 × 10⁵ CFU/g was obtained for raw beef trim, and 2.8 × 10⁵ CFU/sponge was obtained for raw beef brisket (used for beef carcass sponges). The POD was calculated as the number of positive outcomes divided by the total number of trials (12). The POD was calculated for the POD_CP, POD_CC, dPOD_CP, POD_C, POD_R, and dPOD_C. The POD analysis between the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and the SureTect Escherichia coli STEC Identification PCR Assay and the reference methods indicates that there is no significant difference at the 5% level between the number of positive results by the methods at all time points evaluated. The POD analysis between the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and the SureTect Escherichia coli STEC Identification PCR Assay presumptive and confirmed results indicates that there is no significant difference at the 5% level for all methods at all time points following both the traditional confirmation procedure and the alternative confirmation procedure. A summary of POD analyses (13) are presented in Tables  8–11.

Table 8.

Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay results and SureTect Escherichia coli STEC Identification PCR Assay, candidate versus reference—POD results

Matrix	Strain	Time points, ha	MPN/test portionb	n c	Candidate			Reference			${\text{dPOD}}_{\mathrm{C}}$ g	95% CIh
					x d	${\text{POD}}_{\mathrm{C}}$ e	95% CI	x	${\text{POD}}_{\mathrm{R}}$ f	95% CI
Fresh raw spinach (375 g)	E. coli O157:H7 ATCC 51657	8 and 24	NAi	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	–0.43, 0.43
			0.49 (0.25, 0.84)	20	9	0.45	0.26, 0.66	7	0.35	0.18, 0.57	0.10	–0.19, 0.37
			2.62 (1.20, 5.74)	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	–0.43, 0.43
Raw beef trim (375 g)	E. coli O45 MSU DEC11C	8 and 24	NA	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	–0.43, 0.43
			0.55 (0.29, 0.93)	20	10	0.50	0.30, 0.70	8	0.40	0.22, 0.61	0.10	–0.19, 0.37
			1.97 (0.91, 4.27)	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	–0.43, 0.43

^aResults were identical at 8 and 24 h for both PCR assays evaluated.

^bMPN is calculated using the LCF MPN calculator ver. 1.6 provided by AOAC RI, with 95% confidence interval.

^c n = Number of test portions.

^d x = Number of positive test portions.

^ePOD_C = Candidate method presumptive positive outcomes confirmed positive divided by the total number of trials.

^fPOD_R = Reference method confirmed positive outcomes divided by the total number of trials.

^gdPOD_C = Difference between the confirmed candidate method result and reference method confirmed result POD values.

^h95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

ⁱNA = Not applicable.

Table 11.

Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and STEC Screening PCR Assay results and SureTect Escherichia coli STEC Identification PCR Assay, presumptive versus confirmed–—POD results

Matrix	Strain	Time points, ha	CFU/test areab	n c	Presumptive			Confirmedf			${\text{dPOD}}_{\text{CP}}$ h	95% CIi
					xd	${\text{POD}}_{\text{CP}}$ e	95% CI	x	${\text{POD}}_{\text{CC}}$ g	95% CI
Beef carcass sponges	E. coli O111 MSU TW07926	8 and 24	NAj	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	–0.47, 0.47
			58	20	9	0.45	0.26, 0.66	9	0.45	0.26, 0.66	0.00	–0.13, 0.13
			520	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	–0.47, 0.47

^aResults were identical at 8 and 24 h for both PCR assays evaluated.

^bMatrix was treated as an environmental surface.

^c n = Number of test portions.

^dx = Number of positive test portions.

^ePOD_CP = Candidate method presumptive positive outcomes divided by the total number of trials.

^fResults obtained following the alternative confirmation were identical to results obtain from confirmation by USDA/FISIS-MLG 5C.00 reference method.

^gPOD_CC = Candidate method confirmed positive outcomes divided by the total number of trials.

^hdPOD_CP= Difference between the candidate method presumptive result and candidate method confirmed result POD values.

ⁱ95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

^jNA = Not applicable.

Table 9.

Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay results and SureTect Escherichia coli STEC Identification PCR Assay, candidate versus reference—POD results

Matrix	Strain	Time points, ha	CFU/test areab	n c	Candidate			Reference			${\text{dPOD}}_{\mathrm{C}}$ g	95% CIh
					xd	${\text{POD}}_{\mathrm{C}}$ e	95% CI	x	${\text{POD}}_{\mathrm{R}}$ f	95% CI
Beef carcass sponges	E. coli O111 MSU TW07926	8 and 24	NAi	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	–0.43, 0.43
			58	20	9	0.45	0.26, 0.66	7	0.35	0.18, 0.57	0.10	–0.19, 0.37
			520	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	–0.43, 0.43

^aResults were identical at 8 and 24 h for both PCR assays evaluated.

^bMatrix was treated as an environmental surface.

^c n = Number of test portions.

^dx = Number of positive test portions.

^ePOD_C = Candidate method presumptive positive outcomes confirmed positive divided by the total number of trials.

^fPOD_R = Reference method confirmed positive outcomes divided by the total number of trials.

^gdPOD_C = Difference between the confirmed candidate method result and reference method confirmed result POD values.

^h95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

ⁱNA = Not applicable.

Table 10.

Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and STEC Screening PCR Assay results and SureTect Escherichia coli STEC Identification PCR Assay, presumptive versus confirmed—POD results

Matrix	Strain	Time points, ha	MPN/test portionb	n c	Presumptive			Confirmedf			${\text{dPOD}}_{\text{CP}}$ h	95% CIi
					xd	${\text{POD}}_{\text{CP}}$ e	95% CI	x	${\text{POD}}_{\text{CC}}$ g	95% CI
Fresh raw spinach (375 g)	E. coli O157:H7 ATCC 51657	8 and 24	NAj	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	–0.47, 0.47
			0.49 (0.25, 0.84)	20	9	0.45	0.26, 0.66	9	0.45	0.26, 0.66	0.00	–0.13, 0.13
			2.62 (1.20, 5.74)	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	–0.47, 0.47
Raw beef trim (375 g)	E. coli O45 MSU DEC11C	8 and 24	NA	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	–0.47, 0.47
			0.55 (0.29, 0.93)	20	10	0.50	0.30, 0.70	10	0.50	0.30, 0.70	0.00	–0.13, 0.13
			1.97 (0.91, 4.27)	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	–0.47, 0.47

^aResults were identical at 8 and 24 h for both PCR assays evaluated.

^bMPN is calculated using the LCF MPN calculator ver. 1.6 provided by AOAC RI, with 95% confidence interval.

^c n = Number of test portions.

^dx = Number of positive test portions.

^ePOD_CP = Candidate method presumptive positive outcomes divided by the total number of trials.

^fResults obtained following the alternative confirmation were identical to results obtain from confirmation by FDA/BAM Chapter 4A and USDA/FISIS-MLG 5C.00 reference method.

^gPOD_CC = Candidate method confirmed positive outcomes divided by the total number of trials.

^hdPOD_CP = Difference between the candidate method presumptive result and candidate method confirmed result POD values.

ⁱ95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

^jNA = Not applicable.

Robustness Study

For the robustness study the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay workflow was conducted with three testing parameter variations. The three varied parameters included: enrichment time (7, 24, and 26 h), volume of sample lysed (9, 10, and 11 µL), and volume of lysate analyzed (18, 20, and 22 µL). A total of nine combinations of varied parameters were evaluated, with the ninth combination prepared as nominal conditions to provide a comparison. Twenty 375 g test portions of fresh raw spinach were analyzed. Ten were inoculated with E. coli O157:H7 at a level to yield fractional positive results and ten were uninoculated. All 375 g test portions of fresh raw spinach were enriched with 3375 ± 5 mL pre-warmed (41.5 ± 1°C) BPW-ISO and then incubated according to the factorial design. The SureTect Escherichia coli STEC Identification PCR Assay was further evaluated by analyzing an additional twenty 375 g test portions of raw beef trim. Ten raw beef trim test potions were inoculated with E. coli O26 ATCC BAA-1653 at a level to yield fractional positive results and ten were uninoculated. All 375 g test portions of raw beef trim were enriched with 1500 mL pre-warmed (41.5 ± 1°C) mTSB and incubated according to the factorial design. All samples were randomized and tested in a blind-coded fashion.

The data show that the performance of the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay was impacted by small changes in testing parameters at an incubation of 7 h and less than 8 h with fewer positives detected at the 7 h incubation compared to the 8 h. However, consequential POD analysis showed that this observable difference was not statistically significant at the 5% confidence level, but the confidence interval did confirm that more positives were detected at the 8 h timepoint. The SureTect Escherichia coli STEC Identification PCR Assay demonstrated that small changes in testing parameters did not impact the performance of the assay, with POD analysis finding no statistically significant differences between the alternative and nominal conditions at the 5% confidence level. The POD analysis for each target analyte and treatment combination are presented in Tables  12 and 13.

Table 12.

Robustness Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay—POD results

Test combinationa	Test parameters			n b	Test Condition Results			Nominal Conditione Results			${\text{dPOD}}_{\text{TN}}$ g	95% CIh
	Enrichment time, h	Sample volume lysed,  µL	Volume of lysate analyzed,  µL		xc	${\text{POD}}_{\mathrm{T}}$ d	95% CI	x	${\text{POD}}_{\mathrm{N}}$ f	95% CI
Inoculated test portions (E. coli O157:H7 ATCC 43894)
1	7	9	18	10	2	0.20	0.06, 0.51	6	0.6	0.31, 0.83	–0.40	–0.67, 0.02
2	7	9	22	10	1	0.10	0.00, 0.40	6	0.6	0.31, 0.83	–0.50	–0.75, 0.08
3	7	11	18	10	1	0.10	0.00, 0.40	6	0.6	0.31, 0.83	–0.50	–0.75, 0.08
4	7	11	22	10	0	0.00	0.00, 0.28	6	0.6	0.31, 0.83	–0.60	–0.83, 0.20
5	26	9	18	10	6	0.60	0.31, 0.83	6	0.6	0.31, 0.83	0.00	–0.37, 0.37
6	26	9	22	10	6	0.60	0.31, 0.83	6	0.6	0.31, 0.83	0.00	–0.37, 0.37
7	26	11	18	10	6	0.60	0.31, 0.83	6	0.6	0.31, 0.83	0.00	–0.37, 0.37
8	26	11	22	10	6	0.60	0.31, 0.83	6	0.6	0.31, 0.83	0.00	–0.37, 0.37
Uninoculated test portions
1	7	9	18	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
2	7	9	22	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
3	7	11	18	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
4	7	11	22	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
5	26	9	18	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
6	26	9	22	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
7	26	11	18	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
8	26	11	22	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28

^aEach test condition is being compared to the nominal test condition. Note: Test conditions 1–4 (7 h enrichment) and test conditions 5–8 (26 h enrichment) were compared to the nominal condition in different experiments.

^b n = Number of test portions per condition.

^cx = Number of positive test portions per condition.

^dPOD_T = Positive outcomes divided by the total number of trials per condition.

^eNominal condition = 8–24 h enrichment, 10 µL of sample volume lysed, and 20 µL of lysate analyzed.

^fPOD_N = Positive outcomes divided by the total number of trials per nominal condition.

^gdPOD_TN = Difference in POD between the test condition and nominal condition.

^h95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

Table 13.

Thermo Scientific SureTect Escherichia coli STEC Identification PCR Assay robustness study—POD results

Test combinationa	Test parameters			n b	Test condition results			Nominal conditione results			${\text{dPOD}}_{\text{TN}}$ g	95% CIh
	Enrichment time, h	Sample volume lysed,  µL	Volume of lysate analyzed,  µL		xc	${\text{POD}}_{\mathrm{T}}$ d	95% CI	x	${\text{POD}}_{\mathrm{N}}$ f	95% CI
Inoculated test portions (E. coli O26 ATCC BAA-1653)
1	7	9	18	10	9	0.90	0.60, 1.00	9	0.90	0.60, 1.00	0.00	–0.32, 0.32
2	7	9	22	10	9	0.90	0.60, 1.00	9	0.90	0.60, 1.00	0.00	–0.32, 0.32
3	7	11	18	10	9	0.90	0.60, 1.00	9	0.90	0.60, 1.00	0.00	–0.32, 0.32
4	7	11	22	10	9	0.90	0.60, 1.00	9	0.90	0.60, 1.00	0.00	–0.32, 0.32
5	26	9	18	10	9	0.90	0.60, 1.00	9	0.90	0.60, 1.00	0.00	–0.32, 0.32
6	26	9	22	10	9	0.90	0.60, 1.00	9	0.90	0.60, 1.00	0.00	–0.32, 0.32
7	26	11	18	10	9	0.90	0.60, 1.00	9	0.90	0.60, 1.00	0.00	–0.32, 0.32
8	26	11	22	10	9	0.90	0.60, 1.00	9	0.90	0.60, 1.00	0.00	–0.32, 0.32
Uninoculated test portions
1	7	9	18	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
2	7	9	22	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
3	7	11	18	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
4	7	11	22	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
5	26	9	18	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
6	26	9	22	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
7	26	11	18	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28
8	26	11	22	10	0	0.00	0.00, 0.28	0	0.00	0.00, 0.28	0.00	–0.28, 0.28

^aEach test condition is being compared to the nominal test condition. Note: Test conditions 1–4 (7 h enrichment) and test conditions 5–8 (26 h enrichment) were compared to the nominal condition in different experiments.

^b n = Number of test portions per condition.

^cx = Number of positive test portions per condition.

^dPOD_T = Positive outcomes divided by the total number of trials per condition.

^eNominal condition = 8–24 h enrichment, 10 µL sample volume lysed, and 20 µL lysate analyzed.

^fPOD_N = Positive outcomes divided by the total number of trials per nominal condition.

^gdPOD_TN = Difference in POD between the test condition and nominal condition.

^h95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

Matrix Study—ADRIA

The matrix study carried out at ADRIA Développement was conducted adhering to the AOAC guidelines for validation of microbiological methods following the AOAC RI Performance Tested Methods^SM following an unpaired study design for 25 g fresh cut tomatoes, 25 g baby leaves, and 25 g frozen raw beef. The SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC dentification PCR Assay workflow was validated against the internal method from ADRIA Développement, based on ISO/TS 13136 (5).

All three matrixes were artificially contaminated as follows: E. coli O145 Ad983 was used to inoculate 25 g fresh cut tomatoes, E. coli O111 Ad981 was used for 25 g baby leaves, and E. coli O157:H7 Ad683 was used to inoculate 25 g frozen raw beef.

The matrix study consisted of evaluating a total of 30 unpaired 25 g samples for both the candidate and the reference method for all matrixes. Within each sample set, there were 5 uninoculated samples (0 CFU/test portion), 20 low-level inoculated samples (0.2–2 CFU/test portion), and 5 high-level inoculated samples (2–10 CFU/test portion). All test portions were prepared and incubated according to the protocol described in the Sample Preparation section. All samples were analyzed by following the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay at 8 and 24 h time points for all three matrixes. All samples were analyzed using the QuantStudio 5 instrument and results interpreted with the RapidFinder Analysis software. Following the 24 h time point, all samples regardless of presumptive results were culturally confirmed by the ISO 13136 reference method. In addition, the alternative SureTect STEC confirmation procedure was performed as previously described in the Confirmation section.

Internal method from ADRIA Développement, based on ISO/TS 13136—reference method

For the reference method, dilute 25 g of sample (1-in-10) in 225 mL BPW, and then incubate for 18–24 h at 37°C ± 1°C. After incubation, extract sample using the DNeasy Blood and Tissue Kit (Qiagen) in 1 mL enrichment broth (BPW). Take a 5 µL aliquot of sample and run through PCR using the CFX96 from Bio-Rad which will detect the presence of the stx1, stx2, and/or eae genes. After PCR, confirm the sample by directly streaking onto the appropriate selective plate CT-SMAC for presumptive O157:H7 positives, CT-RMAC for presumptive O26 positives, or CHROMagar STEC (for all serogroups). If a pure sample cannot be obtained, perform immunomagnetic separation using Captivate immunobeads.

After streaking onto the appropriate selective agar, incubate for 24 ± 2 h at 37 ± 1°C. Colonies that grow on the plate can then be isolated and confirmed using a latex test, or via PCR on either an isolated colony or 10 pooled colonies.

For the low-spiked samples the contamination level aimed to be 1 CFU/25 g and was 1.2 CFU/25 g for fresh cut tomatoes, 1 CFU/25 g for baby leaves, and 1.4 CFU/25 g frozen raw beef. For the high-spiked samples, the contamination level aimed to be 8 CFU/25 g and was 6.2 CFU/25 g for fresh cut tomatoes, 4.4 CFU/25 g for baby leaves, and 5.9 CFU/25 g frozen raw beef. The contamination level for the frozen raw ground beef matrix was achieved following a seeding protocol with storage at –20°C for 2 weeks. Contamination levels for the baby leaves and fresh cut tomatoes were achieved following a seeding protocol and storage at 5 ± 3°C for 48 h. A total plate count determination on each matrix was performed to ascertain the total microbial load on the day of analysis.

As per criteria outlined in Appendix J of the Official Methods of Analysis Manual, fractional positive results were obtained for 25 g fresh cut tomatoes, 25 g baby leaves, and 25 g frozen raw beef at 8 and 24 h time points for the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and the SureTect Escherichia coli STEC Identification PCR Assay (11). The POD was calculated as the number of positive outcomes divided by the total number of trials (12). The POD was calculated for the POD_CP, POD_CC, dPOD_CP, POD_C, POD_R, and dPOD_C. The POD analysis between the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and the SureTect Escherichia coli STEC Identification PCR Assay and the reference method indicates that there is no significant difference at the 5% level between the number of positive results by the methods at all time points evaluated. The POD analysis between the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and the SureTect Escherichia coli STEC Identification PCR Assay presumptive and confirmed results indicates that there is no significant difference at the 5% level for all methods at all time points following both the traditional confirmation procedure and the alternative confirmation procedure. A summary of POD analyses (13) is presented in Tables  14 and 15.

Table 14.

Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay results, candidate versus reference—POD results

Matrix	Strain	Time points, ha	Contamination levelb	n c	Candidate			Reference			${\text{dPOD}}_{\mathrm{C}}$ g	95% CIh
			CFU/sample		xd	${\text{POD}}_{\mathrm{C}}$ e	95% CI	x	${\text{POD}}_{\mathrm{R}}$ f	95% CI
25 g Frozen ground beef	E. coli O157 Ad683i	8 and 24	NAj	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	–0.43, 0.43
			1.4	20	15	0.75	0.53, 0.89	14	0.70	0.48, 0.85	0.05	–0.22, 0.31
			5.9	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	–0.43, 0.43
25 g Fresh cut tomatoes	E. coli O145 Ad983	8 and 24	N/A	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	–0.43, 0.43
			1.2	20	13	0.65	0.43, 0.82	18	0.90	0.70, 0.97	–0.25	–0.48, 0.01
			6.2	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	–0.43, 0.43
25 g Fresh baby leaves	E. coli O111 Ad981	8 and 24	NA	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	–0.43, 0.43
			1.0	20	4	0.20	0.08, 0.42	5	0.25	0.11, 0.47	–0.05	–0.30, 0.21
			4.4	5	4	0.80	0.38, 1.00	2	0.40	0.12, 0.77	0.40	–0.16, 0.75

^aResults were identical at 8 and 24 h for both PCR assays evaluated.

^bContamination level = CFU level/sample.

^c n = Number of test portions.

^dx = Number of positive test portions.

^ePOD_C = Candidate method presumptive positive outcomes confirmed positive divided by the total number of trials.

^fPOD_R = Reference method confirmed positive outcomes divided by the total number of trials.

^gdPOD_C = Difference between the confirmed candidate method result and reference method confirmed result POD values.

^h95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

ⁱAd = ADRIA Développement, Quimper, France.

^jNA = Not applicable.

Table 15.

Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay, presumptive versus confirmed—POD results

Matrix	Strain	Time points, ha	Contamination levelb	n c	Presumptive			Confirmedf			${\text{dPOD}}_{\text{CP}}$ h	95% CIi
			CFU/sampleb		xd	${\text{POD}}_{\text{CP}}$ e	95% CI	x	${\text{POD}}_{\text{CC}}$ g	95% CI
25 g Frozen ground beef	E. coli O157 Ad683j	8 and 24	NAk	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	–0.47, 0.47
			1.4	20	15	0.75	0.53, 0.89	15	0.75	0.53, 0.89	0.00	–0.13, 0.13
			5.9	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	–0.47, 0.47
25 g Fresh cut tomatoes	E. coli O145 Ad983	8 and 24	NA	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	–0.47, 0.47
			1.2	20	13	0.65	0.43, 0.82	13	0.65	0.43, 0.82	0.00	–0.13, 0.13
			6.2	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	–0.47, 0.47
25 g Fresh baby leaves	E. coli O111 Ad981	8 and 24	NA	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	–0.47, 0.47
			1.0	20	4	0.20	0.08, 0.42	4	0.20	0.08, 0.42	0.00	–0.13, 0.13
			4.4	5	4	0.80	0.38, 1.00	4	0.80	0.38, 1.00	0.00	–0.47, 0.47

^aResults were identical at 8 and 24 h for both PCR assays evaluated.

^bContamination level = CFU level/sample.

^c n = Number of test portions.

^dx = Number of positive test portions.

^ePOD_CP = Candidate method presumptive positive outcomes divided by the total number of trials.

^fConfirmed = Confirmed positives.

^gPOD_CC = Candidate method confirmed positive outcomes divided by the total number of trials.

^hdPOD_CP = Difference between the candidate method presumptive result and candidate method confirmed result POD values.

ⁱ95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

^jAd = ADRIA Développement, Quimper, France.

^kNA = Not applicable.

Results and Discussion

The Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay successfully detected E. coli O157:H7 in fresh raw spinach and frozen raw beef after 8 and 24 h time points. The Thermo Scientific SureTect Escherichia coli O157:H7 STEC Screening PCR Assay and the SureTect Escherichia coli STEC Identification PCR Assay correctly detected E. coli O45 in 375 g raw beef trim along with E. coli O111 in beef carcass sponges after 8 and 24 h time points. Both assays also successfully detected E. coli O145 in 25 g fresh cut tomatoes and E. coli O111 in 25 g baby leaves after 8 and 24 h timepoints. Using POD analysis, no statistically significant differences were observed between the number of positive samples detected by the candidate methods and the reference method for all samples and matrixes tested. The SureTect Escherichia coli STEC Identification PCR Assay correctly excluded E. coli O157:H7 from all samples for the primary enrichments at both 8 and 24 h for the 375 g spinach and 25 g frozen raw beef matrixes. In addition, the SureTect Escherichia coli STEC Identification PCR Assay correctly excluded E. coli O157:H7 from all colonies analyzed for the alternative confirmation.

The detection inclusivity/exclusivity study successfully detected and characterized all 61 inclusivity isolates, with 13 O157:H7 isolates correctly excluded by the SureTect Escherichia coli STEC Identification PCR Assay as expected. None of the 35 exclusivity strains were detected by either kit.

The confirmation inclusivity/exclusivity study demonstrated that the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and the SureTect Escherichia coli STEC Identification PCR Assay successfully confirmed and characterized all 162 inclusivity isolate genotypes, with 25 isolates successfully excluded by the SureTect Escherichia coli STEC Identification PCR Assay as expected from all three selective agars and non-selective agar. The 105 exclusivity isolates were not confirmed by the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay. Of the 105 exclusivity isolates, the SureTect Escherichia coli STEC Identification PCR Assay detected 10 as expected, and successfully excluded the 95 remaining isolates.

As expected, several strains did not recover on the CHROMagar STEC and/or CT-SMAC (see Tables  4 and 5 footnotes for strains that did not recover). This is due to the highly selective nature of the plates. This has been seen previously in a study involving CHROMagar STEC medium in which only 61% of cultured STEC strains grew on the plate (14). There is strong association between the presence of the TerB, TerC, TerD, and TerE genes in STEC isolates (provides resistance to the potent oxidizing agent tellurite which is a component of CHROMagar STEC and also CT-SMAC) and their consequential growth on CHROMagar STEC media. All the 61% of strains that grew during this previous study possessed the TerB gene, so the lack of growth seen on CHROMagar here and also CT-SMAC may be the result of a lack of the Ter gene cluster in certain strains, potentially in combination with the organism's stress levels (14). However, this lack of recovery is not a cause for concern as the problem strains grew on the other tested agars and the user guide instructs that if an isolate fails to grow on the chosen selective agar, to re-culture on one of the other agars, so no STEC isolate is missed/undetected.

The stability study results and consequential POD analysis demonstrate no significant differences at the 5% confidence level, showing that manufacturing and performance are equivalent between kit lots, which demonstrates no overall degradation of the product and supports the shelf-life statement.

For the robustness study, whilst not statistically significant after POD analysis at the 5% confidence level, the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay demonstrated that small changes in testing parameters did impact the performance of the assay at an incubation of 7 h and less than 8 h. The data show, in particular the POD confidence interval, that at an incubation time of 7 h compared to 8 h there were notably fewer positive samples detected. This is not a cause for concern as the user guide stipulates that the minimum incubation time for samples is 8 h, so samples should not be tested at 7 h.

The SureTect Escherichia coli STEC Identification PCR Assay demonstrated that small changes in testing parameters did not impact the performance of the assay, with no statistically significant differences found after POD analysis between the altered conditions and the nominal conditions at the 5% confidence level.

The Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay are robust, quick, and simple to perform, providing results in around 80 min post enrichment. The QuantStudio 5 instrument and software is user-friendly and easy to use. The data generated provides confidence for the end user to utilize the method as both a screening of food samples and confirmation procedure from isolated colonies on selective agar plates.

Conclusions

The data from these studies, within their statistical uncertainty, support the product claims of the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay as a screening and confirmation method for E. coli STEC groups found in 375 g fresh raw spinach, 25 g fresh cut tomatoes, 25 g fresh baby leaves, 25 g frozen raw beef, 375 g raw beef trim, and beef carcass sponges. The POD analysis results for the matrix studies demonstrate that there are no statistically significant differences between the candidate method and any of the reference methods for all samples tested.

The detection inclusivity/exclusivity studies correctly identified all inclusivity isolates and none of the exclusivity isolates, with the SureTect Escherichia coli STEC Identification PCR Assay successfully excluding the anticipated inclusivity isolates.

This was also seen for the more extensive confirmation inclusivity/exclusivity study where all target and non-target analytes were correctly confirmed, characterized, and excluded respectively by the four agar plates validated.

The Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay has shown consistency between kits at different stages of expiry through real-time stability lot-to-lot studies with no statistically significant differences found via POD analysis at the 5% confidence level.

The robustness study demonstrated that for the Thermo Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay only, whilst POD analysis showed that there were no significant differences between the conditions, small changes in testing parameters did impact the performance of the assay at an incubation of 7 h and less than 8 h, with fewer positive samples detected. However, data for the SureTect Escherichia coli STEC Identification PCR Assay demonstrate that small changes in testing parameters did not impact the performance of the assay with no statistical difference between the nominal and alternative conditions at the 5% confidence level.

Acknowledgments

Submitting Company

Oxoid Ltd, part of Thermo Fisher Scientific

Wade Rd

Basingstoke

Hampshire, RG24 8PW, UK

Independent Laboratories

Q Laboratories, Inc.

1400 Harrison Avenue

Cincinnati, OH 45214, USA

ADRIA Développement

20 AV Plage des Gueux Creac h Gwen

29196 Quimper, France

Reviewers

Thomas Hammack

U.S. Food and Drug Administration

Center for Food Safety and Applied Nutrition

5100 Paint Branch Parkway

College Park, MD 20740, USA

Michael Brodsky

Brodsky Consultants

73 Donnamora Crescent

Thornhill

ON L3T 4K6, Canada

Maria Cristina Fernandez

Independent Consultant

Universidad Maimonides

Hidalgo 775, C1405 CABA

Argentina

Conflict of Interest

There were no conflicts of interest from any authors in this paper.