Thermo Fisher Scientific SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay User guide

Brand: Thermo Fisher Scientific

Model: SureTect Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay

Type: User guide

UNI 03/13 - 10/20

http://nf-validation.afnor.org/en

ALTERNATIVE ANALYTICAL METHODS

FOR AGRIBUSINESS

For testing of Food and Environmental samples only.

SureTect™ Escherichia coli O157:H7 and

STEC Screening PCR Assay and SureTect™

Escherichia coli STEC Identiﬁcation PCR

Assay

USER GUIDE

Lysis and real-time PCR detection of Escherichia coli STEC

serotypes in food samples

for use with:

Applied Biosystems™ QuantStudio™ 5 Food Safety Real-Time PCR Instrument

with Thermo Scientiﬁc™ RapidFinder™ Analysis Software v3.0 or later

Catalog NumbersA56838, A56840

Publication NumberMAN0026647

Revision C.0

Revision history:Pub.No.MAN0026647

Revision Date Description

C.0 12 July 2023

•A note was added for instrument dye calibration.

•The RapidFinder™ Analysis Software version was updated.

B.0 21 April 2023

•The RapidFinder™ Analysis Software assay ﬁle names were updated.

•A lysis step for dairy was added.

A.0 8 December 2022

New document for the combined SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay

and SureTect™ Escherichia coli STEC Identiﬁcation PCR Assay User Guide for AOAC and AFNOR. It

supersedes Pub. Nos. MAN0018690 (AOAC) and MAN0018689 (AFNOR).

The changes since the last revisions are as follows:

•New SKUs were created for the kits.

•Kit contents were modiﬁed—the blue dye moved from the Lysis Reagent 1 (LR1) to Proteinase K

(ProK).

•Pierceable lysis seals were added to kit contents.

•New handling tools—cutting tool, capping tool, and uncapping tool—were added to the Lysis

Materials table.

•Improved plate/tube products were added to the PCR Materials table.

•PikoReal™ Real-Time PCR System was removed following equivalence studies approved by

AFNOR Certiﬁcation and AOAC INTERNATIONAL with the 7500 Fast Real-Time PCR System and

QuantStudio™ 5 Real‑Time PCR System.

•The RapidFinder™ Analysis Software version was updated.

The customer is responsible for validation of assays and compliance with regulatory requirements that pertain to their procedures and

uses of the instrument.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE

LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR

ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Trademarks: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed. AOAC is a

trademark and Performance Tested Methods is a service mark of AOAC INTERNATIONAL. AFNOR and NF VALIDATION are trademarks

of Association Française de Normalisation (AFNOR). TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission

and license. CHROMagar is a trademark of Balel S.A. Cy is a registered trademark of GE Healthcare UK Limited.

For testing of Food and Environmental samples only.

Contents

■CHAPTER1Productinformation .................................................. 6

Productdescription ............................................................. 6

Name and intendeduse ..................................................... 6

Principle of the test ......................................................... 6

Procedure overview ......................................................... 7

Limitations ................................................................. 7

Contents and storage ............................................................ 8

Required materials .............................................................. 9

Materials forenrichment ..................................................... 9

Materials forlysis .......................................................... 10

Materials forPCR .......................................................... 11

Materials for conﬁrmation testing ............................................ 12

AOAC Performance Tested MethodsSM validatedworkﬂow ........................... 13

ISO 16140-2:2016 validatedworkﬂow ............................................ 13

■CHAPTER2Before youbegin .................................................... 14

Proceduralguidelines ........................................................... 14

Guidelines for sampleenrichment ............................................ 14

Guidelines for samplelysis .................................................. 14

Guidelines forPCR ........................................................ 15

Guidelines for spinning of PCRtubes ......................................... 15

■CHAPTER3Enrich food or environmental samples – AOAC

Performance Tested MethodsSM .................................................. 16

Enrich foodsamples ............................................................ 16

Enrich production environmentsamples .......................................... 17

■CHAPTER4Enrich food samples - EN ISO16140-2:2016 .................... 18

Enrich foodsamples ............................................................ 18

SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

■CHAPTER5Prepare the lysate using the thermal cyclermethod ............. 20

■CHAPTER6Perform PCR with the ScreeningAssay .......................... 22

PCR with the QuantStudio™ 5 Food Safety Real-Time PCR Instrument and

RapidFinder™ Analysis Software v3.0 or later .................................... 22

Set up the plate layout in RapidFinder™ Analysis Software ...................... 22

Set up the PCR reactions ................................................... 22

Load and run the reactions .................................................. 23

View results and dataanalysis ............................................... 24

■CHAPTER7Perform PCR with the IdentiﬁcationAssay ....................... 25

PCR with the QuantStudio™ 5 Food Safety Real-Time PCR Instrument and

RapidFinder™ Analysis Software v3.0 or later .................................... 25

Set up the plate layout in RapidFinder™ Analysis Software ...................... 25

Set up the PCR reactions ................................................... 25

Load and run the reactions .................................................. 26

View results and dataanalysis ............................................... 27

■CHAPTER8Conﬁrm positive results - AOAC Performance Tested

MethodsSM and EN ISO 16140-2:2016 .......................................... 28

Recommended conﬁrmation methods ........................................... 28

Isolate presumptivepositives .................................................... 28

Conﬁrm presumptive positives - AOAC Performance Tested MethodsSM and

NF VALIDATION™............................................................ 30

■APPENDIXATroubleshooting .................................................... 31

Test control organisms .......................................................... 33

■APPENDIXBCalibrate the Cy® 5.5 dye for SureTect™ assayworkﬂows ..... 34

Perform a background calibration ................................................ 34

Thaw, vortex, and centrifuge the calibration plate .................................. 34

Add the dye to the software ..................................................... 35

Perform the dye calibration ...................................................... 35

View calibration results ......................................................... 36

■APPENDIXCSupplementalinformation ........................................ 37

AOAC Performance Tested MethodsSMCertiﬁcation ................................. 37

ISO 16140-2:2016 according to NF VALIDATION™ by AFNORCertiﬁcation ............ 38

Good laboratory practices forPCR ............................................... 40

Contents

4SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Description of materials for lysis andPCR ......................................... 40

Symboldeﬁnitions ............................................................. 44

■APPENDIXDSafety ............................................................... 45

Chemicalsafety ................................................................ 46

Biological hazardsafety ......................................................... 47

■APPENDIXEDocumentation and support ...................................... 48

Food safety support ............................................................ 48

Customer and technical support ................................................. 48

Relateddocumentation ......................................................... 49

References ................................................................................ 50

Contents

SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Product information

IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix

in this document.

Product description

Name and intended use

The Thermo Scientiﬁc™ SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and

SureTect™ Escherichia coli STEC Identiﬁcation PCR Assay are part of a complete workﬂow for the rapid

presence/absence detection of Escherichia coli O157:H7 and the Escherichia coli STEC serogroups

(O26, O45, O103, O111, O121, and O145) from food samples. These assays are for use in laboratories

undertaking microbiological analysis. See Table1 for compatible instruments and software.

Note: The qPCR instrument must be calibrated with the following dyes before use: FAM™, VIC™, ABY™,

JUN™, Cy5™, and Cy5.5™.

Table1Instruments and software

PCR Instrument Software Assay type Pathogen Assay File[1]

Applied Biosystems™

QuantStudio™ 5 Food

Safety Real-Time PCR

Instrument

Thermo Scientiﬁc™

RapidFinder™ Analysis

Software v3.0 or later

Screening E.coli-STEC-Screen-ST-A56838-

QS5-1.2 or later

Identiﬁcation E.coli-STEC-ID-ST-A56840-QS5-1.2

or later

[1] Assay files and instructions are available at thermofisher.com/molecular-microbiology-software.

Principle of the test

This assay is based on TaqMan™ PCR technology. Dye-labeled probes target unique DNA sequences

speciﬁc to Escherichia coli STEC serotypes, and an internal positive control (IPC). Target DNA, if

present, is detected by real-time PCR. Analysis software provides interpretation of results. For more

information about real-time PCR, go to thermoﬁsher.com/qpcreducation.

The IPC template, primers, and probe provide an internal control with each reaction to show that the

PCR process has occurred. It is unnecessary to incorporate positive control organisms with routine

testing of samples.

6SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Procedure overview

Enriched food samples are combined directly with ready-to-use Lysis Reagent 1 and Proteinase K, to

lyse bacterial cells present in the sample and release their DNA into solution.

Lysates are transferred to the Escherichia coli O157:H7 and STEC Screening PCR Tubes or the

Escherichia coli STEC Identiﬁcation PCR Tubes to rehydrate the lyophilized PCR pellets. The pellets

contain lyophilized target-speciﬁc primers, dye-labelled probes, and PCR master mix components. The

PCR tubes are sealed, loaded into the real-time PCR instrument, then the run is started using the

RapidFinder™ Analysis Software. After the run is complete, the software displays the interpreted results

as simple positive or negative symbols. The results can be reported, stored, printed, and downloaded

as required.

Results are achieved approximately 80 minutes after loading the prepared sample into the instrument.

See Figure1 for the manual workﬂow.

Figure1Manual workﬂow

Limitations

•The test is designed to detect DNA from target organisms that have been present at a minimum

level of 1 CFU/sample, and have grown to detectable levels during the enrichment.

•The customer is responsible for validation of sample matrices or culture media not described in this

document.

•When testing a sample type or culture medium that has not been validated, we recommend testing

a selection of known negative and positive samples, to ensure that expected results are achieved.

See “Test control organisms” on page33 and ENISO22174:2005.

•See Appendix A, “Troubleshooting” for additional information.

Chapter1Product information

Product description 1

SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Contents and storage

Store the kits protected from light, at 2–8°C. Bring to room temperature (23±5°C) before opening.

Table2SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay, 96 tests (Cat.

No.A56838)

Contents Amount Storage[1,2]

Lysis Reagent 1 Tubes (clear liquid containing ﬁne

white particles) 12strips of 8 tubes

2–8°C

Thermo Scientiﬁc™ 96-Well Pierceable Seals 2 sheets

Proteinase K (blue liquid) 1tube

Escherichia coli O157:H7 and STEC Screening PCR

Tubes

12 strips of 8 tubes

1 pellet each

PCR Caps 12 strips of 8 caps

[1] Store the kit protected from light.

[2] Opening the kit does not affect shelf life.

Table3SureTect™ Escherichia coli STEC Identiﬁcation PCR Assay, 96 tests (Cat. No. A56840)

Contents Amount Storage[1,2]

Escherichia coli STEC Identiﬁcation PCR Tubes 12 strips of 8 tubes

1 pellet each 2–8°C

PCR Caps 12 strips of 8 caps

[1] Store the kit protected from light.

[2] Opening the kit does not affect shelf life.

Chapter1Product information

Contents and storage

8SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Required materials

Unless otherwise indicated, all materials are available through the Thermo Fisher Microbiology ordering

process or thermoﬁsher.com. MLS: Fisher Scientiﬁc (ﬁsherscientiﬁc.com) or other major laboratory

supplier.

Catalog numbers that appear as links open the web pages for those products.

Note: Parts may ship separately depending on conﬁguration and storage conditions.

Materials for enrichment

Table4Equipment, accessories, and consumables

Item Source

Homogenizer laboratory blender or diluter, one of the following or equivalent:

•Homogenizer Laboratory Blender

•Diluﬂux™ Pro Automated Gravimetric Dilutor with

simple (non-robotic) dispensing arm

•Diluﬂux™ Pro Automated Gravimetric Dilutor with

robotic dispensing arm

DB5000A

DB4100A

DB4150A

Sample enrichment bags, one of the following or equivalent:

•BagFilter™ 400 (400 mL)

•BagPage™ 400 (400 mL)

•BagLight™ 400 (400 mL)

•RollBag™ 1300 (1300 mL)

DB4011A

DB4012A

DB4013A

DB4014A

Incubator ﬁtted with racks for homogenizer bags thermoﬁsher.com

Disposable gloves MLS

Variable volume single-channel pipette, 1‑ to 10‑mL

Available through the Thermo Fisher Microbiology

ordering process.

96-well rack

Filtered pipette tips, 1‑ to 10‑mL

Sample tubes, 1.5‑mL

Table5Media

Item Source

Oxoid™ Buered Peptone Water (ISO) CM1049B or equivalent

Oxoid™ Modiﬁed Tryptone Soya Broth CM0989B or equivalent

Acriﬂavine MLS

Chapter1Product information

Required materials 1

SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Materials for lysis

For description of materials, see page40.

Table6Materials for lysis of enriched cultures

Item Source

Plastics, consumables, and reagents

Single-channel pipette, 10‑ to 100‑µL

Electronic adjustable spacing, multichannel pipette,

10‑ to 100‑µL

Available through the Thermo Fisher Microbiology

ordering process.

Single-channel stepper pipette, 10‑ to 100‑µL

Filtered pipette tips, 10‑ to 100‑µL

Domed Lysis Tube Caps A56895

Thermo Scientiﬁc™ 96-Well Pierceable Seals A55331

MicroAmp™ Adhesive Film Applicator

(for pierceable seals) 4333183

Thermo Scientiﬁc™ PCR Strip Cutting Tool A55329

Thermo Scientiﬁc™ PCR Capping Tool A55327

Thermo Scientiﬁc™ PCR Uncapping Tool A55328

Additional materials for the thermal cycler method

Applied Biosystems™ SimpliAmp™ Thermal Cycler A24811

SimpliAmp™ Strip Plate Tray A55333

Thermo Scientiﬁc™ 96-Well PCR Base A55330

Chapter1Product information

Required materials

10 SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Materials for PCR

For description of materials, see page40.

Table7Materials for PCR

Item Source

QuantStudio™ 5 Food Safety Real-Time PCR

Instrument, 0.1-mL block, with RapidFinder™

Analysis Software v3.0 or later

•SureTect™ Escherichia coli O157:H7 and STEC

Screening PCR Assay and Pathogen Assay

File: E.coli-STEC-Screen-ST-A56838-QS5-1.2

or later

•SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay and Pathogen Assay File: E.coli-

STEC-ID-ST-A56840-QS5-1.2 or later

A36320 (desktop)

A36328 (laptop)

Contact your local microbiology sales representative

Additional materials for PCR

Vortex mixer

Available through the Thermo Fisher Microbiology

ordering process. See thermoﬁsher.com/plastics

for more information.

Plate centrifuge

8-channel pipette, 10‑ to 100‑µL

Filtered pipette tips, 10‑ to 100‑µL

For the QuantStudio™ 5 Food Safety Real-Time PCR Instrument

Applied Biosystems™ QuantStudio™ 5 Spectral

Calibration Plate (Cy® 5.5 dye), 96‑well, 0.1mL A45218

SimpliAmp™ Strip Plate Tray Cover (optional) A55971

QuantStudio™ 5 Strip Plate Tray A55332

Thermo Scientiﬁc™ 96-Well PCR Base A55330

Thermo Scientiﬁc™ PCR Strip Cutting Tool A55329

Thermo Scientiﬁc™ PCR Capping Tool A55327

Thermo Scientiﬁc™ PCR Uncapping Tool A55328

Chapter1Product information

Required materials 1

SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Materials for conﬁrmation testing

Table8Materials for conﬁrmation of positive results

Item Source

Oxoid™ Sorbitol MacConkey Agar CM0813B or equivalent

Oxoid™ C-T Supplement SR0172E or equivalent

Escherichia coli O157 Latex Test DR0620M

Remel™ RIM E. coli O157:H7 Latex Test R24250

Chromogenic Coliform Agar (Dehydrated) CM1205B

Dynabeads™ anti-E. coli O157, 1mL 71003

Dynabeads™ EPEC/VTECO145, 2mL 71007

Dynabeads™ MAX EPEC/VTECO45Kit, 100 reactions A14631

Dynabeads™ MAX EPEC/VTECO121Kit, 100 reactions A14632

Dynabeads™ EPEC/VTECO111, 2mL 71009

Dynabeads™ EPEC/VTECO26, 2mL 71013

Dynabeads™ EPEC/VTECO103, 2mL 71011

Commercially available latex agglutination kits for serogroups O26,

O103, O111, O145 MLS

Chapter1Product information

Required materials

12 SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

AOAC Performance Tested MethodsSM validated workﬂow

Enrich food samples (page16)

▼

Prepare the lysate using the thermal cycler method (page20)

▼

Perform PCR with the Screening Assay

(page22)

Perform PCR with the Identiﬁcation Assay

(page25)

▼

Conﬁrm positive results - AOAC Performance Tested MethodsSM and EN ISO 16140-2:2016

(page28)

ISO 16140-2:2016 validated workﬂow

Enrich food samples (page18)

▼

Prepare the lysate using the thermal cycler method (page20)

▼

Perform PCR with the Screening Assay

(page22)

▼Perform PCR with the Identiﬁcation Assay

(page25)

Conﬁrm positive results - AOAC Performance Tested MethodsSM and EN ISO 16140-2:2016

(page28)

Chapter1Product information

AOAC Performance Tested MethodsSM validated workﬂow 1

SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Before you begin

Procedural guidelines

Guidelines for sample enrichment

•For preparation of initial suspensions, follow the instructions of EN ISO6887 and EN ISO16654

standards. Comply with Good Laboratory Practices (refer to EN ISO 7218:2007 standard).

•Follow the manufacturer's instructions for preparation of culture media.

•When following the short enrichment protocol, if pre-warming is applicable, ensure that the

enrichment broth is pre-warmed for 18–24 hours before adding to the sample.

•Use ﬁltered homogenizer bags to help with fat and particle separation.

•For consistent PCR results, use a ventilated incubator.

•Follow the speciﬁed temperature allowances.

•Dispose of all inoculated culture media as hazardous microbiological waste, even if shown to be

negative for the target organism, according to local guidelines.

Guidelines for sample lysis

•Ensure that you use the recommended materials (see “Description of materials for lysis and PCR”

on page40).

•Follow the speciﬁed temperature allowances.

•For downstream PCR on the QuantStudio™ 5 Instrument —Prepare a mock-puriﬁed sample, using

sterile enrichment media instead of enriched sample, as a negative extraction control. (The negative

extraction control is optional but recommended for RapidFinder™ Analysis Software.)

Add the enriched sample or negative extraction control to the bottom of the lysis tube.

•For the thermal cycler method, in order to prevent crushing tubes, use one of the following:

–Use the SimpliAmp™ Strip Plate Tray.

Note: When the SimpliAmp™ Strip Plate Tray is used, the detachable side-edges of the 96-

well lysis plate must always be broken o and discarded before placing the strips into the tray.

Note: When running a full 96-well lysis plate in the SimpliAmp™ instrument, the SimpliAmp™

Strip Plate Tray is not required.

–Balance at least 4 complete tube strips in the sample block. We recommend spacing the

strips evenly across the sample block, and if needed, add empty SureTect™ tubes to make 4

complete strips.

14 SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Guidelines for PCR

•Ensure that you use the recommended materials (see “Description of materials for lysis and PCR”

on page40).

•PCR pellets are pale yellow. If the pellet is collapsed or not pale yellow, do not use.

•IMPORTANT! After the lysate has been added to the pellets, vortex and spin the pellets and

ensure that the pellets rehydrate immediately by tapping the tubes on the lab bench. Start the PCR

run within 30 minutes.

•Tube and cap strips can be cut when less than a full strip is required.

Do not cut the strips of caps or tubes too close to the wall of the tube or the cap lid, otherwise the

lid might not seal adequately during PCR.

•After the PCR tubes have been opened, add lysate within 10 minutes.

•Particulate matter from the lysate can inhibit the PCR.

To ensure that no particles are transferred from the

Lysis Reagent 1 Tube to the PCR tube, remove lysate

from the top half of the liquid, taking care not to disrupt

the particles at the bottom of the tube.

If the particles become disturbed, allow the particles to

resettle for 1–2 minutes before lysate removal.

•Ensure that the pellet is fully dissolved. The solution

changes from blue to green when the pellet is

dissolved.

•For ease of use, a multi-channel pipettor can be used

to transfer multiple lysates to the PCR tubes.

•Follow “Good laboratory practices for PCR” on

page40. For more information go to www.thermoﬁsher.com/us/en/home/life-science/pcr/

real-time-learning-center/real-time-pcr-basics.html.

Guidelines for spinning of PCR tubes

•SureTect™ workﬂow:

–20 μL of SureTect™ lysate is added to each PCR tube.

–The PCR tubes are vortexed for 10–15 seconds to ensure that the pellet is fully rehydrated.

–User must ensure that the reaction mixture is at the bottom of the PCR tube.

•A rapid spin-down is highly recommended before the PCR run to:

– Collect the reaction mixture at the bottom of the well.

– Remove bubbles.

•This ensures that the reaction conditions are optimal, and as a result, the PCR step is less likely to

fail or to suer from unwanted signal ﬂuctuations which could aect the interpretation.

•Centrifugation of PCR tubes is included in every GLP (Good Laboratory Practice) protocol.

Figure2Avoid lysis particles

Chapter2Before you begin

Procedural guidelines 2

SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Enrich food or environmental

samples – AOAC Performance

Tested MethodsSM

Enrich food samples

In this procedure, refer to Table9 for media volumes and incubation times.

1. Pre-warm the indicated volume of media to 41.5±1°C.

2. Transfer the food sample to a homogenizer bag, then add the pre-warmed media, as indicated.

Table9Enrichment conditions

Matrices PCR instrument Media Incubation

Up to 375 g raw leafy

produces (e.g. fresh raw

spinach) except fresh

herbs (e.g. cilantro or

parsley)

QuantStudio™ 5

Instrument and

RapidFinder™ Analysis

Software v3.0 or later

1-in-10 ratio of sample

in pre-warmed Buered

Peptone Water (ISO)[1]

41.5±1°C for

8‑24hours

Up to 375 g beef trim

375 g of sample and 1500

mL of prewarmed Modiﬁed

Tryptone Soya Broth

Beef carcass sponge

1x sponge and 200 mL

of pre-warmed Modiﬁed

Tryptone Soya Broth

Up to 25 g raw beef meat

(e.g. frozen raw ground

beef)

1-in-10 ratio of sample

in pre-warmed Buered

Peptone Water (ISO)[2]

Up to 25 g raw leafy

produces (e.g. fresh baby

leaves)

Up to 25 g raw

vegetables and fruits (e.g.

fresh cut tomatoes)

[1] For example, 375g of sample in 3375mL of pre-warmed media.

[2] For example, 25g of sample and 225mL of pre-warmed media.

16 SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

3. Homogenize the sample.

•For soft samples—homogenize for 30 seconds to 1 minute using a homogenizer.

•For samples containing hard particles, such as bone—squeeze the bag by hand until the

sample is mixed thoroughly with the media.

4. Incubate as described in Table9.

5. Remove the enriched sample from the incubator, brieﬂy mix the liquid in the homogenizer bag/tube

by hand, transfer an aliquot of sample from the ﬁltered side of the bag to a new tube, then close

the tube and brieﬂy mix.

Retain sucient sample for conﬁrmation or repeat testing.

Proceed directly to Prepare the lysate using the thermal cycler method (page20), or store the retained

sample at 2–8°C for a maximum of 72 hours. Do not exceed 72 hours of total storage time.

Enrich production environment samples

For all samples, pre-warm the indicated volume of media to 41.5±1°C.

1. Transfer the sample to a homogenizer bag or suitable screw capped vessel, then add the media, as

indicated.

Table10Environmental sample enrichment conditions

Sample type PCR instrument Media Incubation

Beef carcass sponge

QuantStudio™ 5 Instrument

and RapidFinder™ Analysis

Software v3.0 or later

1x sponge and 200 mL

of pre-warmed Modiﬁed

Tryptone Soya Broth

41.5±1°C for 8–

24hours

2. Homogenize thoroughly by hand.

3. Before incubation, roll down the tops of the enrichment bags to minimize the headspace of air

contained in the bag. For liquids, use sterile screw cap tubes with < 5mm of headspace after the

sample is added.

4. Incubate as described in Table10.

5. Remove the enriched sample from the incubator, mix gently, then transfer an aliquot of sample to a

new tube, and close the tube. Retain sucient sample for conﬁrmation of repeat testing.

Proceed directly to Prepare the lysate using the thermal cycler method (page20), or store the retained

sample at 2–8°C for a maximum of 72 hours. Do not exceed 72 hours of total storage time.

Chapter3Enrich food or environmental samples – AOAC Performance Tested MethodsSM

Enrich production environment samples 3

SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Enrich food samples - EN ISO

16140-2:2016

Enrich food samples

1. Pre-warm the indicated volume of media to 41.5±1°C.

2. Transfer the food sample to a homogenizer bag, then add the media, as indicated.

Table11Enrichment conditions

Matrices PCR instrument Media Incubation

375 g raw meat (excluding

poultry)

QuantStudio™ 5

Food Safety Real-

Time PCR Instrument

and RapidFinder™

Analysis Software

v3.0 or later

1-in-5 ratio of sample in

pre-warmed Buered Peptone

Water (ISO)[1]

41.5±1°C for

8–24hours

375 g raw vegetables and

fruits and juices (excluding

sprouted seeds)

1-in-10 ratio of sample in

pre-warmed Buered Peptone

Water (ISO)[2]

41.5±1°C for

10–24hours

25 g raw meat (excluding

poultry)

25 g raw vegetables and

fruits and juices

1-in-10 ratio of sample in

pre-warmed Buered Peptone

Water (ISO)[3]

41.5±1°C for

8–24hours

25 g dairy

1-in-10 ratio of sample in

Buered Peptone Water (ISO)

supplemented with 5mg/L

acriﬂavine[4]

41.5±1°C for

16–20 hours

[1] For example, 375g of sample in 1500mL of pre-warmed media.

[2] For example, 375g of sample in 3375mL of pre-warmed media.

[3] For example, 25g of sample in 225mL of pre-warmed media.

[4] For example, 25g of sample in 225mL of media.

3. Homogenize the sample.

•For soft samples—homogenize for 30 seconds to 1 minute using a homogenizer.

•For samples containing hard particles, such as bone—squeeze the bag by hand until the

sample is mixed thoroughly with the media.

18 SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

4. Incubate as described in Table11.

5. Remove the enriched sample from the incubator, brieﬂy mix the liquid in the homogenizer bag/tube

by hand, transfer an aliquot of sample from the ﬁltered side of the bag to a new tube, then close

the tube and brieﬂy mix.

Retain sucient sample for conﬁrmation or repeat testing.

Proceed directly to Prepare the lysate using the thermal cycler method (page20), or store the retained

sample at 2–8°C for a maximum of 72 hours. Do not exceed 72 hours of total storage time.

Chapter4Enrich food samples - EN ISO 16140-2:2016

Enrich food samples 4

SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

Prepare the lysate using the thermal

cycler method

Before you begin, ensure that you are using the recommended materials (see “Description of materials

for lysis and PCR” on page40).

Note: Always wear clean gloves when handling pierceable seals and store pierceable seals in the kit

box or in an enclosed bag to prevent contamination of the pierceable seal.

1. Equilibrate the Lysis Reagent 1 Tubes to room temperature (23±5°C).

a. Place the required number of Lysis Reagent 1 Tubes in a Thermo Scientiﬁc™ 96-Well PCR

Base and SimpliAmp™ Strip Plate Tray. Use the Thermo Scientiﬁc™ PCR Strip Cutting Tool if

less than full strips are needed.

b. Ensure the liquid is collected at the bottom of each tube—ﬂick the tubes in a downward

motion or tap the tubes against the bench.

c. Allow the tubes to remain at room temperature (23±5°C) for approximately 10 minutes before

opening.

2. Remove the foil from each Lysis Reagent 1 Tube, then add 10µL of Proteinase K to the tube.

These tubes are referred to as Lysis Tubes in the rest of the procedure.

IMPORTANT! Avoid contamination of the Proteinase K stock tube. Use a new ﬁltered pipette tip

each time Proteinase K is withdrawn from the stock tube. Use a 10–100 µL stepper pipette to

reduce the number of tips required.

3. For raw dairy samples only, dilute the enriched sample 1-in-2. For example, 1mL of enrichment

sample and 1mL of Buered Peptone Water.

4. Transfer 10 µL of the enriched sample to a Lysis Tube. For the negative extraction controls, transfer

10 µL of sterile enrichment media to a Lysis Tube.

Ensure that the pipette tip reaches the bottom of the Lysis Tube to facilitate complete mixing of the

sample with the lysis reagents.

20 SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identiﬁcation

PCR Assay User Guide

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