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

  • Hello! I've reviewed the user guide for the Thermo Fisher Scientific SureTect Escherichia coli O157:H7 PCR Assay, which details the use of QuantStudio 5 and 7500 Fast Real-Time PCR instruments with RapidFinder software for detecting E. coli O157:H7 in food samples. This document outlines procedures for sample enrichment, lysis, and PCR, along with key features like real-time detection and internal controls. I'm ready to assist you with any specific questions about the procedures, compatible materials, or troubleshooting.
  • What is the purpose of the SureTect Escherichia coli O157:H7 PCR Assay?
    Which instruments and software are compatible with this assay?
    What kind of samples can be tested with this kit?
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.
©2023 Thermo Fisher Scientific Inc. All rights reserved.
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 MethodsSMCertification ................................. 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.
1
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
1
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
or
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
1
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)
or
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)
or
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
1
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:
2
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.
or
–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
2
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.
3
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.
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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
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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").
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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
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20 SureTect™ Escherichia coli O157:H7 PCR Assay User Guide
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