Thermo Fisher Scientific SureTect Escherichia coli O157:H7 PCR Assay User guide

Brand: Thermo Fisher Scientific

Model: SureTect Escherichia coli O157:H7 PCR Assay

Type: User guide

UNI 03/10 - 03/15

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

ALTERNATIVE ANALYTICAL METHODS

FOR AGRIBUSINESS

LICENSE NUMBER 021501

For testing of Food and Environmental samples only.

SureTect™ Escherichia coli O157:H7 PCR

Assay

USER GUIDE

Lysis and real-time PCR detection of Escherichia coli O157:H7 in

food samples

for use with:

Applied Biosystems™ QuantStudio™ 5 Real‑Time PCR Instrument with Thermo

Scientiﬁc™ RapidFinder™ Analysis Software v3.0 or later

Applied Biosystems™ 7500 Fast Real‑Time PCR Instrument with Applied

Biosystems™ RapidFinder™ Express Software v2.0 or later

Catalog NumberA56844

Publication NumberMAN0026680

RevisionD.0

Revision history:Pub.No.MAN0026680

Revision Date Description

D.0 7 July 2023

•A note was added for instrument dye calibration.

•The RapidFinder™ Analysis Software version was updated.

C.0 3 March 2023

•The RapidFinder™ Analysis Software and RapidFinder™ Express Software assay ﬁle names were

updated.

•The NF VALIDATION™ by AFNOR Certiﬁcation verbiage was updated.

B.0 27 January 2023

•The footnote for the AOAC's Validated matrices table was revised to include raw beef trim.

•The AFNOR validated matrices were updated to include frozen vegetables and raw beef meat

product types.

A.0 8 December 2022

New document for the combined SureTect™ Escherichia coli O157:H7 PCR Assay User Guide for AOAC

and AFNOR. It supersedes Pub. Nos. MAN0017803 (AOAC) and MAN0017804 (AFNOR).

The changes since the last revisions are as follows:

•New SKU was created for the kit.

•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.

•A new chapter was added, Conﬁrm presumptive colonies using SureTect™ Escherichia coli O157:H7

PCR Assay - EN ISO 7218:2007 (page30).

•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 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.

Limited Use Label License: This product or portions hereof are subject to proprietary rights of Epoch Biosciences, Inc. and are made

and sold under license from Epoch under the patents and patent applications as may be designated by Epoch from time to time set

forth, including one or more of the following: U.S. Patent Nos. 6,699,975, 7,718,374, 7,348,146 and 7,671,218, and related U.S. and

foreign patents and pending applications. A license must be obtained directly from Epoch with respect to any other proposed use of this

product.

Buyer agrees and understands it is not licensed to (a) provide commercial data or databases to or on behalf of any third party that relate

to use of the product; or (b) use the product or data there from in a clinical diagnostic setting where data from an individual's sample is

given to such individual or their caregiver. Neither Epoch Biosciences nor its licensors grants any other licenses, express or implied, to

permit the manufacture, use, sale, or importation of the product, any other nucleic acids, or any data generated from use of the product.

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). Windows is a trademark of Microsoft. Solaris is a trademark of Dharmacon Inc.

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 ............................................................ 7

Required materials .............................................................. 8

Materials forenrichment ..................................................... 8

Materials forlysis ........................................................... 9

Materials for PCR ......................................................... 10

Materials for conﬁrmation testing ............................................ 11

AOAC Performance Tested MethodsSM validatedworkﬂow ........................... 12

ISO 16140-2:2016 validatedworkﬂow ............................................ 12

■CHAPTER2Before youbegin .................................................... 13

Proceduralguidelines ........................................................... 13

Guidelines for sampleenrichment ............................................ 13

Guidelines for samplelysis .................................................. 13

Guidelines forPCR ........................................................ 14

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

■CHAPTER3Enrich food samples – AOAC Performance Tested MethodsSM . 16

Enrich foodsamples ............................................................ 16

■CHAPTER4Enrich food samples - EN ISO16140-2:2016 .................... 17

Enrich food samples—raw beef meat and fruits andvegetables ...................... 17

■CHAPTER5Prepare the lysate ................................................... 19

Prepare the lysate using the thermal cyclermethod ................................ 19

Prepare the lysate using the heat blockmethod .................................... 21

SureTect™ Escherichia coli O157:H7 PCR Assay User Guide 3

■CHAPTER6PerformPCR ........................................................ 22

PCR with the QuantStudio™ 5 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 ............................................... 23

PCR with the 7500 Fast Instrument and RapidFinder™ Express Software v2.0 or later ... 25

Set up the platelayout ..................................................... 25

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

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

View results and dataanalysis ............................................... 27

■CHAPTER7Conﬁrm SureTect™ E.coli 0157:H7 positive results - AOAC

Performance Tested MethodsSM and EN ISO 16140-2:2016 .................. 28

Recommended conﬁrmation methods ........................................... 28

Isolate presumptive positives and conﬁrm SureTect™ E.coli0157:H7 ................. 28

■CHAPTER8Conﬁrm presumptive colonies using SureTect™

Escherichia coli O157:H7 PCR Assay - EN ISO 7218:2007 .................. 30

Conﬁrm characteristic colonies using SureTect™ Escherichia coli O157:H7 PCRAssay .. 30

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

Test control organisms .......................................................... 32

RapidFinder™ Express Software resultswarnings .................................. 33

■APPENDIXBSupplementalinformation ........................................ 34

AOAC Performance Tested MethodsSMCertiﬁcation ................................. 34

NF VALIDATION™ by AFNORcertiﬁcation ......................................... 36

Good laboratory practices forPCR ............................................... 38

Description of materials for lysis andPCR ......................................... 38

Symboldeﬁnitions ............................................................. 41

■APPENDIXCSafety ............................................................... 42

Chemicalsafety ................................................................ 43

Biological hazardsafety ......................................................... 44

Contents

4SureTect™ Escherichia coli O157:H7 PCR Assay User Guide

■APPENDIXDDocumentation and support ...................................... 45

Food safety support ............................................................ 45

Customer and technical support ................................................. 45

Relateddocumentation ......................................................... 46

References ................................................................................ 47

Contents

SureTect™ Escherichia coli O157:H7 PCR Assay User Guide 5

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 PCR Assay enables real-time PCR

detection of Escherichia coli O157:H7 from food samples. This kit is 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: VIC™, JUN™, and

Cy5™.

Table1Instruments and software

PCR instrument Software Pathogen Assay File

Applied Biosystems™

QuantStudio™ 5 Real‑Time PCR

Instrument

Thermo Scientiﬁc™ RapidFinder™

Analysis Software v3.0 or later

E.coli O157H7-ST-A56844-QS5-2.1

or later[1]

Applied Biosystems™ 7500 Fast

Real‑Time PCR Instrument

Applied Biosystems™

RapidFinder™ Express Software

v2.0 or later

E.coliO157H7_SureTect_7500_2.0 or

later[1]

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

Principle of the test

This assay is based upon use of Solaris™ reagents for performing PCR. Dye-labeled probes target

unique DNA sequences speciﬁc to Escherichia coli O157:H7 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 assay includes an internal positive control for each reaction to conﬁrm that the PCR process has

occurred. It is unnecessary to incorporate positive control organisms with routine testing of samples.

6SureTect™ Escherichia coli O157:H7 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 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™ 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.

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32 and ENISO22174:2005.

•For more information, see Appendix A, “Troubleshooting”.

Contents and storage

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

Table2SureTect™ Escherichia coli O157:H7 PCR Assay, 96 tests (Cat. No.A56844)

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 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.

Chapter1Product information

Contents and storage 1

SureTect™ Escherichia coli O157:H7 PCR Assay User Guide 7

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3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4Media

Item Source

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

Oxoid™ Modiﬁed Tryptone Soya Broth CM0989B or equivalent

Chapter1Product information

Required materials

8SureTect™ Escherichia coli O157:H7 PCR Assay User Guide

Materials for lysis

For description of materials, see page38.

Table5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 heat block method[1]

Heat block

MLS

Timer

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

[1] The heat block method is not compatible with the 96-well Pierceable Seals. The pierceable seals provided with the kit must not be

used, and it is mandatory to purchase domed caps separately.

Chapter1Product information

Required materials 1

SureTect™ Escherichia coli O157:H7 PCR Assay User Guide 9

Materials for PCR

For description of materials, see page38.

Table6Materials for PCR

Item Source

Real-time PCR instrument and accessories, one of the following instrument packages

QuantStudio™ 5 Real‑Time PCR Instrument, 0.1-mL

block, with RapidFinder™ Analysis Software v3.0 or

later

For use with SureTect™ Escherichia coli O157:H7

PCR Assay and Pathogen Assay File: E.coli

O157H7-ST-A56844-QS5-2.1 or later

A36320 (desktop) or equivalent

A36328 (laptop) or equivalent

Contact your local microbiology sales representative

7500 Fast Real‑Time PCR Instrument with

RapidFinder™ Express Software v2.0 or later

For use with SureTect™ Escherichia coli

O157:H7 PCR Assay and Pathogen Assay File:

E.coliO157H7_SureTect_7500_2.0 or later

A30304 (desktop)

A30299 (laptop)

Contact your local microbiology sales representative

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

QuantStudio™ 5 Strip Plate Tray A55332

Thermo Scientiﬁc™ 96-Well PCR Base A55330

For the 7500 Fast Food Safety Real-Time PCR Instrument

Thermo Scientiﬁc™ 96-Well PCR Base A55330

7500 Fast 96W Strip Plate Adaptor A46347

Armadillo™ Low-Proﬁle PCR Strip Plate, 96-well,

clear, pack of 5[1] AB2696SMP

Ultra Clear qPCR Caps, strips of 8 PT0615

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

SimpliAmp™ Strip Plate Tray Cover (optional) A55971

Thermo Scientiﬁc™ PCR Strip Cutting Tool A55329

Thermo Scientiﬁc™ PCR Capping Tool A55327

[1] Used as balancing tubes and managing the lid pressure if less than 2 full strips are processed.

Chapter1Product information

Required materials

10 SureTect™ Escherichia coli O157:H7 PCR Assay User Guide

Materials for conﬁrmation testing

Table7Materials for conﬁrmation of positive results

Item Source

Oxoid™ Sorbitol MacConkey Agar CM0813B or equivalent

Oxoid™ C-T Supplement SR0172E or equivalent

Dynabeads™ anti-E. coli O157 71003

Dry-Bags™ Maximum Recovery Diluent (MRD) DB0733W

Escherichia coli O157 Latex Test DR0620M

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

Oxoid™ Novobiocin Selective Supplement SR0181E or equivalent

Modiﬁed Tryptone Soya Broth (Dehydrated) (mTSB) CM0989 or equivalent

Chapter1Product information

Required materials 1

SureTect™ Escherichia coli O157:H7 PCR Assay User Guide 11

AOAC Performance Tested MethodsSM validated workﬂow

Enrich food samples (page16)

▼



Prepare the lysate using the thermal cycler

method (page19) or Prepare the lysate using the heat block

method (page21)

▼

PCR with the QuantStudio™ 5 Instrument

and RapidFinder™ Analysis Software v3.0

or later (page22)

PCR with the 7500 Fast Instrument and

RapidFinder™ Express Software v2.0 or

later (page25)

▼

Isolate presumptive positives and conﬁrm SureTect™ E.coli 0157:H7 (page28)

ISO 16140-2:2016 validated workﬂow

Enrich food samples—raw beef meat and fruits and vegetables (page17)

▼

Prepare the lysate using the thermal cycler

method (page19) or Prepare the lysate using the heat block

method (page21)

▼

PCR with the QuantStudio™ 5 Instrument and

RapidFinder™ Analysis Software v3.0 or later

(page22)

PCR with the 7500 Fast Instrument and

RapidFinder™ Express Software v2.0 or later

(page25)

▼

Isolate presumptive positives and conﬁrm SureTect™ E.coli 0157:H7 (page28)

Chapter1Product information

AOAC Performance Tested MethodsSM validated workﬂow

12 SureTect™ Escherichia coli O157:H7 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.

•The short protocols of detection are sensitive to conditions of incubation. It is required to respect

scrupulously the conditions of temperature indicated in the technical speciﬁcation. Notably, you

must verify that the temperature or pre-warming of the enrichment broth reaches the required

temperature. The time of preparation of the samples, delay between the end of the step of pre-

warming of the enrichment broth, and the beginning of the step of incubation of the food samples,

must not exceed 45 minutes. Using a ventilated incubator during incubation is recommended.

•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38).

•Follow the speciﬁed temperature allowances.

•For downstream PCR on the 7500 Fast instrument or the QuantStudio™ 5 Instrument —Prepare

a mock-puriﬁed sample using sterile enrichment media as a negative extraction control. (The

negative extraction control is required for RapidFinder™ Express Software; it 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:

SureTect™ Escherichia coli O157:H7 PCR Assay User Guide 13

–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.

Guidelines for PCR

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

on page38).

•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38. For more information go to www.thermoﬁsher.com/us/en/home/life-science/pcr/

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

Figure1Avoid lysis particles

Chapter2Before you begin

Procedural guidelines

14 SureTect™ Escherichia coli O157:H7 PCR Assay User Guide

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.

Chapter2Before you begin

Procedural guidelines 2

SureTect™ Escherichia coli O157:H7 PCR Assay User Guide 15

Enrich food samples – AOAC

Performance Tested MethodsSM

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 pre-warmed media, as indicated.

Table8Enrichment conditions

Matrices PCR instrument Media Incubation

375 g Raw ground

beef

•QuantStudio™

5 Instrument

and RapidFinder™

Analysis Software

v2.0 or later

•7500 Fast Instrument

and RapidFinder™

Express Software

v2.0 or later

1-in-4 ratio of sample in

pre-warmed Oxoid™ Modiﬁed

Tryptone Soya Broth[1,2]

41.5±1°C for 9–24

hours

375g Raw beef trim 1-in-5 ratio of sample in

pre-warmed Oxoid™ Modiﬁed

Tryptone Soya Broth[3,1]

41.5°C for 9–24

hours

25g Apple juice

25 g Fresh spinach

1-in-10 ratio of sample in

pre-warmed Oxoid™ Modiﬁed

Tryptone Soya Broth[4]

41.5±1°C for 8–24

hours

[1] For example, 375g of sample to 1,125mL of media.

[2] Choose the 1-in-4 or 1-in-5 ratio based on your test requirements.

[3] For example, 375g of sample to 1,500mL of media.

[4] For example, 25g sample to 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.

4. Incubate as described in Table8.

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 Chapter 5, “Prepare the lysate”, or store the retained sample at 2–8°C for a

maximum of 72 hours. Do not exceed 72 hours of total storage time.

16 SureTect™ Escherichia coli O157:H7 PCR Assay User Guide

Enrich food samples - EN ISO

16140-2:2016

Enrich food samples—raw beef meat and fruits and

vegetables

1. For the raw beef meat samples only, 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9Enrichment conditions

Matrices PCR instrument Media Incubation

25 g Raw beef meat

products

•Fresh and frozen

ground beef

•Seasoned beef

•Fresh and frozen

beef trim

•QuantStudio™ 5

Instrument and

RapidFinder™ Analysis

Software v3.0 or later

•7500 Fast Instrument

and RapidFinder™

Express Software v2.0

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

25 g Fruits and

vegetables

•Sprouts

•Leafy greens

•Frozen

vegetables

•Fruits and juices

1-in-10 ratio of sample in

Buered Peptone Water

(ISO)[1]

41.5±1°C for 20‑24

hours

[1] For example, 25g of sample to 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.

SureTect™ Escherichia coli O157:H7 PCR Assay User Guide 17

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 Chapter 5, “Prepare the lysate”, 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—raw beef meat and fruits and vegetables

18 SureTect™ Escherichia coli O157:H7 PCR Assay User Guide

Prepare the lysate

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38).

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. 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 Lysis Reagent 1.

4. Close or seal the tubes with the domed Lysis Tube Caps using the Thermo Scientiﬁc™ PCR

Capping Tool or Thermo Scientiﬁc™ 96-Well Pierceable Seals using the MicroAmp™ Adhesive Film

Applicator.

Note: Samples sealed with domed Lysis Tube Caps and samples sealed with pierceable ﬁlm can

be placed together in the same run on the SimpliAmp.

IMPORTANT! To prevent crushing the tubes in the SimpliAmp™ Thermal Cycler, use the

SimpliAmp™ Strip Plate Tray, or use a full 96-well plate (see "Guidelines for sample lysis").

SureTect™ Escherichia coli O157:H7 PCR Assay User Guide 19

Note: Whenever 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.

There is no need to break o the side-edges of the strip plate if a full 96-well plate is used without

the tray. When running a full 96-well lysis plate in the SimpliAmp instrument, the SimpliAmp™ Strip

Plate Tray is not required.

Note: The SimpliAmp™ Strip Plate Tray Cover should not be used in the lysis step in the

SimpliAmp™ Thermal Cycler.

5. Incubate the samples in the SimpliAmp™ Thermal Cycler using the following program settings.

Table10Program settings

Step Temperature Time

1 37°C 10 minutes

2 95°C 5 minutes

3 10°C 2 minutes

4 4°C Hold[1]

[1] For convenience, samples can be held at 4°C until proceeding to PCR or transfer to storage at 2–8°C.

6. Ensure that the lid heater is on and set to 105°C, and the volume is set to Maximum.

7. Carefully remove the Lysis Tubes from the SimpliAmp™ Thermal Cycler. Do not agitate the Lysis

Tubes.

8. Proceed to Chapter 6, “Perform PCR”.

(Optional) Store the samples at 2–8°C for up to 24 hours, including any time stored at 4°C in the thermal

cycler.

Note: If pierceable seals have been used and already pierced, a second seal can be used for re-sealing

the pierced tube before storing the samples.

Chapter5Prepare the lysate

Prepare the lysate using the thermal cycler method

20 SureTect™ Escherichia coli O157:H7 PCR Assay User Guide

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Thermo Fisher Scientific SureTect Escherichia coli O157:H7 PCR Assay User guide

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Model: SureTect Escherichia coli O157:H7 PCR Assay

Type: User guide

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Thermo Fisher Scientific SureTect Escherichia coli O157:H7 PCR Assay User guide

Thermo Fisher Scientific SureTect Escherichia coli O157:H7 PCR Assay User guide

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