Thermo Fisher Scientific SureTect User Manual

SureTect™ Campylobacter jejuni, C. coli and
C. lari PCR Assay

USER GUIDE

Lysis and real-time PCR detection of Campylobacter jejuni,
C. coli, and C. lari in food and environmental samples

for use with:
Applied Biosystems™ QuantStudio™ 5 Food Safety Real-Time PCR Instrument

with Thermo Scientific™ RapidFinder™ Analysis Software v1.1 or later
Applied Biosystems™ 7500 Fast Food Safety Real-Time PCR Instrument with

Applied Biosystems™ RapidFinder™ Express Software v2.0 or later

Catalog Number A44251

Publication Number MAN0018692

Revision B.0

For testing of Food and Environmental samples only.

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.

Revision history: Pub. No. MAN0018692

Revision Date Description

B.0 5 February 2021 Update of the instrument software versioning.

A.0 29 January 2020 New document

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. Windows is a trademark of Microsoft.
TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license.

©2021 Thermo Fisher Scientific Inc. All rights reserved.

Contents

■

CHAPTER 1 Product information .................................................. 5

Product description ............................................................. 5

Name and intended use ..................................................... 5

Principle of the test ......................................................... 5

Procedure overview ......................................................... 6

Limitations ................................................................. 6

Contents and storage ............................................................ 6

Required materials .............................................................. 7

Materials for enrichment ..................................................... 7

Materials for lysis ........................................................... 8

Materials for PCR .......................................................... 9

Materials for confirmation testing ............................................ 10

Workflows .................................................................... 10

■

CHAPTER 2 Before you begin .................................................... 11

Procedural guidelines ........................................................... 11

Guidelines for sample enrichment ............................................ 11

Guidelines for sample lysis .................................................. 11

Guidelines for PCR ........................................................ 11

■

CHAPTER 3 Enrich food samples ................................................ 13

Enrich food samples ............................................................ 13

■

CHAPTER 4 Prepare the lysate ................................................... 15

Prepare the lysate using the thermal cycler method ................................ 15

SureTect

™

Campylobacter jejuni, C. coli and C. lari PCR Assay User Guide—AOAC

3

Contents

■

■

CHAPTER 5 Perform PCR ........................................................ 17

PCR with the QuantStudio™ 5 Instrument and RapidFinder™ Analysis Software

v1.1 or later ................................................................. 17

Set up the plate layout in RapidFinder™ Analysis Software ...................... 17

Set up the PCR reactions ................................................... 17

Load and run the reactions .................................................. 18

View results and data analysis ............................................... 18

PCR with the 7500 Fast Instrument and RapidFinder™ Express Software v2.0 or later ... 20

Set up the plate layout ..................................................... 20

Set up the PCR reactions ................................................... 20

Load and run the reactions .................................................. 21

View results and data analysis ............................................... 22

CHAPTER 6 Confirm positive results ............................................ 23

Recommended confirmation methods ........................................... 23

Isolate presumptive positives .................................................... 23

Confirmation in case of co-infection .............................................. 23

■

APPENDIX A Troubleshooting .................................................... 24

Test control organisms .......................................................... 26

RapidFinder™ Express Software results warnings .................................. 26

■

APPENDIX B Supplemental information ........................................ 27

AOAC Performance Tested MethodsSM Certification ................................. 27

Good laboratory practices for PCR ............................................... 28

Symbol definitions ............................................................. 30

■

APPENDIX C Safety ............................................................... 31

Chemical safety ................................................................ 32

Biological hazard safety ......................................................... 33

■

Documentation and support ....................................................... 34

Food Safety support ............................................................ 34

Related documentation ......................................................... 34

References ................................................................................ 35

4

SureTect™ Campylobacter jejuni, C. coli and C. lari PCR Assay User Guide—AOAC

1

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™ Campylobacter jejuni, C. coli and C. lari PCR Assay enables real-time
PCR detection of Campylobacter jejuni, C. coli, and C. lari from food and environmental samples. This
kit is for use in laboratories undertaking microbiological analysis, and they are compatible with the
following instruments and software:

Product information

PCR instrument

Applied Biosystems
QuantStudio™ 5 Food Safety Real­Time PCR Instrument

Applied Biosystems™ 7500 Fast
Food Safety Real-Time PCR
Instrument

[1]

or equivalents manufactured by Thermo Fisher Scientific and/or subsidiaries.

[2]

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

[1]

™

Thermo Scientific™ RapidFinder
Analysis Software v1.1 or later

Applied Biosystems™ RapidFinder
Express Software v2.0 or later

Software Pathogen Assay File

™

™

Principle of the test

This assay is based on TaqMan™ PCR technology. Dye-labeled probes target unique DNA sequences
specific to Campylobacter jejuni, C. coli, and C. lari, 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.

Campylobacter Multiplex SureTect QS5

version 1.0 or later

C.jejuni Multiplex SureTect 1.0 or later

[2]

[2]

SureTect™ Campylobacter jejuni, C. coli and C. lari PCR Assay User Guide—AOAC

5

Chapter 1 Product information

1

Contents and storage

Procedure overview

Enriched food or environmental 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 SureTect™ Campylobacter jejuni, C. coli and C. lari PCR Tubes to
rehydrate the lyophilized PCR pellets. The pellets contain lyophilized target-specific primers, dye­labelled 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 26 and EN ISO 22174:2005.

•

See Appendix A, “Troubleshooting” for additional information.

Contents and storage

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

Table 1 SureTect™ Campylobacter jejuni, C. coli and C. lari PCR Assay, 96 tests (Cat. No. A44251)

Contents

Lysis Reagent 1 Tubes (clear, pale blue liquid containing fine white
particles)

Lysis Tube Caps, domed 12 strips of 8 caps

Proteinase K (clear colorless liquid) 1 tube

SureTect™ Campylobacter jejuni, C. coli and C. lari PCR Tubes 12 strips of 8 tubes

PCR Caps 12 strips of 8 caps

Amount

12 strips of 8 tubes

1 pellet each

6

SureTect™ Campylobacter jejuni, C. coli and C. lari PCR Assay User Guide—AOAC

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.

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

Materials for enrichment

Table 2 Equipment, accessories, and consumables

Item Source

Chapter 1 Product information

Required materials

1

Homogenizer Laboratory Blender or Dilutor, one of
the following, or equivalent

Homogenizer bags appropriate for the sample type
and size

Incubator fitted with racks for homogenizer bags, set
to 42±1°C

Disposable gloves MLS

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

96-well rack

Filtered pipette tips, 1‑ to 10‑mL

Sample tubes, 1.5‑mL

Available through the Thermo Fisher Microbiology

For DB4100A or DB4150A:

DB5000A

DB4100A

DB4150A

DB4011A

DB4012A

DB4013A

DB4014A

thermofisher.com

ordering process.

Table 3 Media

Item

Bolton Broth, 500 g of base CM0983B

Oxoid™ Modied Bolton Broth Selective Supplement SR0208E

Buered Peptone Water (BPW) MLS

SureTect™ Campylobacter jejuni, C. coli and C. lari PCR Assay User Guide—AOAC

Source

7

Chapter 1 Product information

1

Required materials

Materials for lysis

Table 4 Materials for lysis of enriched cultures

Plastics, consumables, and reagents

Single-channel pipette, 10‑ to 100‑µL

or

Electronic adjustable spacing, multichannel pipette,
10‑ to 100‑µL

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

Filtered pipette tips, 10‑ to 100‑µL

Tool for capping and decapping

Applied Biosystems™ SimpliAmp™ Thermal Cycler A24811

MicroAmp™ 96-Well Tray/Retainer Set for Veriti
Systems

Item Source

Available through the Thermo Fisher Microbiology

ordering process.

™

4381850

MicroAmp™ Splash-Free 96-Well Base 4312063

8

SureTect™ Campylobacter jejuni, C. coli and C. lari PCR Assay User Guide—AOAC

Materials for PCR

Table 5 Materials for PCR

Chapter 1

Product information

Required materials

Item Source

1

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

QuantStudio™ 5 Food Safety Real-Time PCR Instrument, 0.1-mL block, with
RapidFinder™ Analysis Software v1.1 or later

For use with SureTect™ Campylobacter jejuni, C. coli and C. lari PCR
Assay and Pathogen Assay File: Campylobacter Multiplex SureTect QS5

Contact your local microbiology

version 1.0 or later

7500 Fast Food Safety Real-Time PCR Instrument with RapidFinder

™

Express Software v2.0 or later

For use with SureTect™ Campylobacter jejuni, C. coli and C. lari PCR Assay
and Pathogen Assay File: C.jejuni Multiplex SureTect 1.0 or later

Contact your local microbiology

[1]

A36320 (desktop)

A36328 (laptop)

sales representative

A30304 (desktop)

A30299 (laptop)

sales representative

Additional materials for PCR

Vortex mixer

8-channel pipette, 10‑ to 100‑µL

Filtered pipette tips, 10‑ to 100‑µL

Available through the Thermo Fisher

Microbiology ordering process. See

thermofisher.com/plastics for more

information.

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

MicroAmp™ 96-Well Tray for VeriFlex™ Block 4379983

MicroAmp™ Splash-Free 96-Well Base 4312063

For the 7500 Fast Food Safety Real-Time PCR Instrument

Precision Plate holder for SureTect™ assays

or

7500 Fast Precision Plate Holder, for 0.1 mL tube strips

PT0690

or

A29252

PCR Carry plate for SureTect™ assays PT0695

If using Precision Plate holder for SureTect™ assays (Cat. No. PT0690):

VersiPlate PCR Strip Tube Plate, 96-well, low profile

Ultra Clear qPCR Caps, strips of 8

[2]

[2]

AB1800

AB0866

If using 7500 Fast Precision Plate Holder, for 0.1 mL tube strips (Cat. No. A29252):

MicroAmp™ Fast 8-Tube Strip, 0.1 mL

MicroAmp™ Optical 8-Cap Strips

[1]

or equivalents manufactured by Thermo Fisher Scientific and/or subsidaries.

[2]

Used for balancing.

[3]

Required to balance the lid pressure if less than 2 full strips are processed.

[3]

[3]

4358293

4323032

SureTect™ Campylobacter jejuni, C. coli and C. lari PCR Assay User Guide—AOAC

9

Chapter 1 Product information

1

Workflows

Materials for confirmation testing

Table 6 Materials for confirmation of positive results

Item Source

Oxoid™ Campylobacter Blood-Free Selective Agar
Base (Dehydrated), 500 g

Oxoid™ CCDA Selective Supplement, 10 freeze-dried
vials

Remel™ Campy Cefex Agar R110138 (USA)

Brilliance™ CampyCount Agar R110168, PO1185A (Europe)

Oxoid™ O.B.I.S. campy Kit ID0800M

Workflows

“PCR with the QuantStudio™ 5 Instrument

and RapidFinder™ Analysis Software v1.1 or

Enrich food samples (page 13)

▼

Prepare the lysate using the thermal cycler method (page 15)

▼

PCR with the 7500 Fast Instrument and

RapidFinder™ Express Software v2.0 or later

later” on page 17

CM0739B

SR0155E

(page 20)

10

▼

Confirm positive results (page 23)

SureTect™ Campylobacter jejuni, C. coli and C. lari PCR Assay User Guide—AOAC

2

Procedural guidelines

Guidelines for sample enrichment

•

For preparation of master suspensions, follow the instructions of EN ISO 6887 and EN ISO 16654
standards. Comply with Good Laboratory Practices (refer to EN ISO 7218 standard).

•

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

•

Use non-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.

Before you begin

Guidelines for sample lysis

•

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 — To prevent crushing tubes, use the tray only from the
MicroAmp™ 96-Well Tray/Retainer Set provided with the SimpliAmp™ Thermal Cycler. See the
SimpliAmp™ Thermal Cycler User Guide (Pub. No. MAN0009889). Alternatively, use at least 4
complete tube strips in the heat block. We recommend spacing the strips evenly across the heat
block. If needed, add empty SureTect™ tubes to make 4 complete strips.

Guidelines for PCR

•

IMPORTANT! After the lysate has been added to the pellets, ensure that the pellet rehydrates

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.

SureTect™ Campylobacter jejuni, C. coli and C. lari PCR Assay User Guide—AOAC

11