Real-time PCR Detection of Salmonella spp. in Food and · PDF file · 2013-12-13For testing of Food and Environmental samples only. ... MagMAX™ Express-96 Magnetic Particle Processor

USER GUIDE

For testing of Food and Environmental samples only.

Real-time PCR Detection of Salmonella spp.in Food and Environmental SamplesPerformance Tested Methods

SM

-certified workflowfor use with:PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental TestingMagMAX™ Express-96 Magnetic Particle ProcessorMicroSEQ® Salmonella spp. Detection KitApplied Biosystems® 7500 Fast Real-Time PCR InstrumentRapidFinder™ Express Software

Publication Number 4405968

Revision D

For testing of Food and Environmental samples only.

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

DISCLAIMER

LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED ORIMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TOTHE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT,WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIALDAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

LIMITED USE LABEL LICENSE No. 492: Environmental Testing, Quality Control/Quality Assurance Testing, Food and Agricultural Testing

Notice to Purchaser: The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of theproduct (a) to perform internal research for the sole benefit of the purchaser; and (b) for environmental testing, quality control/quality assurancetesting, food and agricultural testing, including reporting results of purchaser's activities in environmental testing, quality control/quality assurancetesting, food and agricultural testing for a fee or other commercial consideration. No other right is hereby granted expressly, by implication, or byestoppel. This product is for environmental testing, quality control/ quality assurance testing, food and agricultural testing and research purposesonly.

The purchase of this product does not grant the purchaser any additional rights, including (without limitation) the right to transfer or resell theproduct in any form or the right to use the product as a therapeutic agent or diagnostics test component. For information on obtaining additionalrights, please contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

Trademarks

The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. AOAC is aregistered trademark and Performance Tested Methods is a service mark of AOAC International. Stomacher is a registered trademark of SewardLimited, UK. Whirl-Pak is a registered trademark of Aristotle Corporation. TaqMan is a registered trademark of Roche Molecular Systems, Inc., usedunder permission and license.

© 2013 Life Technologies Corporation. All rights reserved.

mailto: [email protected]

Contents

About this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

■ CHAPTER 1 Overview .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

■ CHAPTER 2 Enrich food and environmental samples . . . . . . . . . . . . . . . . 7

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

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Enrich Salmonella spp. in buffered peptone water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

■ CHAPTER 3 Isolate DNA with the PrepSEQ® Nucleic AcidExtraction Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Kit contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Required materials not included with the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Procedure overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Before first use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Prepare the reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Before each use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Resuspend Magnetic Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Centrifuge the enriched culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13For large sample pellets: perform the preclarification protocol . . . . . . . . . . . . . . . . . 14For high-fat samples: remove fat layer before lysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Set up the Lysis Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Set up the MagMAX™ Express-96 processing plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Process sample on MagMAX™ Express-96 Magnetic Particle Processor . . . . . . . . . . . . . . . 15

■ CHAPTER 4 PCR with the MicroSEQ® Salmonella spp. DetectionKit and RapidFinder™ Express Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Real-time PCR Detection of Salmonella spp. in Food and Environmental Samples User Guide 3

Kit contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Required materials not included with the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Important procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Avoid fat layer and particulates after sample lysis (collection of DNA sample forPCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Create or edit a run file in RapidFinder™ Express Software . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Prepare the assay beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Set up the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Load and run the reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

View results and data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22If necessary, investigate results in SDS Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

■ CHAPTER 5 Recommended confirmation methods . . . . . . . . . . . . . . . . . 24

■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

■ APPENDIX B Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Kit sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Kit specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Operational conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

AOAC® Performance Tested MethodsSM

Certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Good laboratory practices for PCR/RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

■ APPENDIX C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Specific chemical handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

■ Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Obtaining Certificates of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Food Safety support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

References ......................................................................................... 37

Contents

4 Real-time PCR Detection of Salmonella spp. in Food and Environmental Samples User Guide

About this guide

IMPORTANT! Before using the products described in this guide, read andunderstand the information in the "Safety" appendix in this document.

Revision history

Revision Date Description

D Nov 2013 • Added instructions for PCR with the MicroSEQ® Salmonella spp. Detection Kit andconfirmation testing.

• Changed document title to reflect additional instructions.

• Added logos and certification details for AOAC® Performance Tested MethodsSM

certification.

• Added additional troubleshooting tips.

• Clarified instructions for handling Magnetic Particles, enriching samples in achocolate matrix, and storage of DNA samples in the Elution Plate.

• Updated number format (time, temperature, and centrifugation speeds) for AOAC®

certification.

• Updated to a Life Technologies template with associated updates to the limitedlicense information, warranty information, and trademark statement.

C June 2010 Corrected typographical error in the "Exclusions, Conditions, Exceptions, andLimitations" section.

B September2009

• Added instructions for handling Magnetic Particles, high-fat samples, andcarryover of Magnetic Particles of food particulate residues in final eluates.

• Updated limited license information, warranty information, and trademarkstatements.

A January 2009 New document.


Overview

This guide describes the following AOAC® Performance Tested MethodsSM workflowfor detection of Salmonella spp. in food and environmental samples:

1. Enrichment of 25-g or 25-mL samples in Buffered Peptone Water (BPW).Chocolate-based samples require BPW with skim milk powder and BrilliantGreen dye.

2. Automated preparation of PCR-ready DNA using the PrepSEQ® Nucleic AcidExtraction Kit for Food and Environmental Testing and the MagMAX™

Express-96 Magnetic Particle Processor.The MagMAX™ Express-96 Magnetic Particle Processor enables high-throughputsample processing in a 96-well format with minimal handling.

3. Real-time PCR detection of Salmonella spp. DNA using the MicroSEQ® Salmonellaspp. Detection Kit and RapidFinder™ Express Software on the AppliedBiosystems® 7500 Fast Real-Time PCR Instrument.

4. Confirmation testing of positive samples.

See “AOAC Performance Tested Methods Certification” on page 30 for detailedinformation about the AOAC® certification.

This workflow is intended for use by microbiological analysts who need to test forSalmonella spp. in food or environmental samples. These kits are for use in food andenvironmental testing only. Not for any animal or human therapeutic or diagnosticuse.

Visit www.lifetechnologies.com/foodsafety for a list of workflows for detection ofSalmonella spp.

1


http://www.lifetechnologies.com/foodsafety

Enrich food and environmentalsamples

Required materials

MLS: Major laboratory supplier

Item Source

D/E Neutralizing Broth BD # 281910 or equivalent

Buffered Peptone Water (BPW) MLS

Skim milk powder (for chocolate samples only) MLS

Brilliant Green Dye Solution (for chocolate samples only) MLS

Swabs, cotton MLS

Homogenizer (Stomacher® 400 Laboratory Blender orequivalent)

Seward # 0400/001/AJ orequivalent

Sterile 15-mL conical tubes (for environmental swabsamples)

MLS

Homogenizer bag, as required by sample type:

With sponge, 4.5” × 9” (Whirl-Pak® Speci-SpongeEnvironmental Sampling Bag, or equivalent)

Nasco # B01245WA orequivalent

With mesh, 6” × 9”, 24 oz (Whirl-Pak® Filter Bag forHomogenizer Blender, or equivalent)


No mesh, 6” × 9”, 24 oz (Whirl-Pak® Write-on Bag, orequivalent)


2


Workflow

Enrich food samplesEnrich environmental samples

Using a swab Using a sponge

Homogenize 25 g (25 mL) of foodsample + 225 mL of BPW, then

incubate at 37±1°C, 16–20 hours.

q

Prewet the swab with 0.5 mL of D/ENeutralizing Broth.

q

Wipe the surface area to be testedand add the swab to 10 mL of BPW.

q

Twirl the swab for 90±30 seconds andincubate at 37±1°C, 16–20 hours.

q

Prewet the sponge with 10 mL of D/ENeutralizing Broth.

q

Wipe the surface area to be testedand add the sponge to 100 mL of

BPW.

q

Homogenize the sample and incubateat 37±1°C, 16–20 hours.

q

Proceed to isolate DNA

Enrich Salmonella spp. in buffered peptone water

IMPORTANT! Use proper aseptic technique while handling samples to avoid cross-contamination.

1. Prepare Buffered Peptone Water (BPW) according to the manufacturer’sinstructions.

Note: For growth of bacteria in a chocolate matrix, prepare BPW containing100 g/L of sterile skim milk powder, which reduces the growth inhibitioncharacteristic of chocolate.

2. Combine BPW and sample as described in the following table.

Sample type Method

Food Add 225 mL of BPW (+ skim milk powder for chocolatesamples) to 25 g (or 25 mL) of sample.

Environmental swab 1. Prewet the swab with 0.5 mL of D/E NeutralizingBroth.

2. Wipe the surface area to be tested.

3. Add the swab to 10 mL of BPW (+ skim milk powderfor chocolate samples) in a 15-mL conical tube.

Environmental sponge 1. Prewet the sponge with 10 mL of D/E NeutralizingBroth.

2. Wipe the surface area to be tested.

3. Add the sponge to 100 mL of BPW (+ skim milkpowder for chocolate samples).

Chapter 2 Enrich food and environmental samplesWorkflow2


3. Mix sample with BPW as described in the following table:A filtered bag may be used for enrichment of samples with particulates.

Sample type Method

Food:

• Coarse foodtypes, such asmeat, poultry, andseafood[1]

• Eggs

Process for 1–2 minutes in a homogenizer (Stomacher®

400 Laboratory Blender with speed setting Norm, orequivalent).

Food:Liquids or powdered

Shake the bag at least 25 times to achieve a homogeneoussuspension.

Environmental swab Twirl the swab for 90±30 seconds.

Environmental sponge Use one of the following methods:

• Use a nonfiltered, nonmesh stomacher bag andhomogenize for about 1 minute (Stomacher® 400Laboratory Blender with speed setting Norm, orequivalent)

• Hand squeeze for about 1 minute.

[1] Hand massage foods that cannot be processed in a homogenizer.

4. Incubate at 37±1°C under static conditions for 16–20 hours.

Note: For growth of bacteria in a chocolate matrix:1. Incubate at 37±1°C for 1–2 hours.2. Add Brilliant Green Dye Solution to a final concentration of 0.018 g/L.3. Continue to incubate at 37±1°C for a total of 16–20 hours.

Chapter 2 Enrich food and environmental samplesEnrich Salmonella spp. in buffered peptone water 2


Isolate DNA with the PrepSEQ®

Nucleic Acid Extraction Kit

Product description

The PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing (Cat.nos. 4480466, 4428176) is designed for preparation of high-quality microbial nucleicacid from broth cultures. Magnetic beads allow efficient DNA capture and samplewashing. The kit enables high-throughput automation in a 96-well plate format on theMagMAX™ Express-96 Magnetic Particle Processor. Visit www.lifetechnologies.com/foodsafety for a list of workflows with non-automated DNA isolation methods.

Kit contents and storage

Table 1 PrepSEQ® Nucleic Acid Extraction Kit

ComponentsCat. no.4480466

(100 reactions)

Cat. no.4428176

(300 reactions)Storage[1]

Magnetic Particles 2 × 1.5 mL 6 × 1.5 mL 5±3°C

Lysis Buffer 2 × 50 mL 6 × 50 mL

Roomtemperature(23±5°C)

Binding Solution (Isopropanol)[2] 1 empty bottle 3 empty bottles

Wash Buffer Concentrate[3] 2 × 26 mL 6 × 26 mL

Elution Buffer 1 × 25 mL 3 × 25 mL

Proteinase K (PK) Buffer 1 × 50 mL 3 × 50 mL

Proteinase K, 20 mg/mL 1 × 1.25 mL 3 × 1.25 mL Below –18°C

[1] Refer to the product label for expiration date.[2] See “Required materials not included with the kit” on page 11.[3] Add 74 mL of 95% ethanol before use.

Note: Kit components may ship separately depending on configuration and storageconditions.

3




Required materials not included with the kit

Unless otherwise indicated, all materials are available from Life Technologies. MLS:Major laboratory supplier.

Item Source

Equipment

Benchtop microcentrifuge Eppendorf 5415 D or equivalent

MagMAX™ Express-96 Deep Well Magnetic ParticleProcessor

Cat. no. 4400079

96-Well Magnetic-Ring Stand Cat. no. AM10050

Block heater, 37°C MLS

Laboratory mixer, Vortex or equivalent MLS

Pipettors:

• Positive-displacement

• Air-displacement

• Multichannel

MLS

(Optional but recommended) Plate centrifuge MLS

Consumables

Disposable gloves MLS

Micropipette tips, aerosol-resistant MLS

MagMAX™ Express-96 Deep Well Plates Cat. no. 4388476

MagMAX™ Express-96 Standard Plates Cat. no. 4388475

MagMAX™ Express-96 Deep Well Tip Combs Cat. no. 4388487

(Optional) MicroAmp® Clear Adhesive Film Cat. no. 4306311

Microcentrifuge tubes, PCR clean, 1.5-mL MLS

Sterile tubes, 15-mL or 50-mL MLS

Reagents

Ethanol, 95% MLS

Isopropanol, 100% MLS

Nuclease-free Water Cat. no. AM9938

Chapter 3 Isolate DNA with the PrepSEQ® Nucleic Acid Extraction KitRequired materials not included with the kit 3


Procedure overview

1 mL of enriched sample is centrifuged. If the enriched sample produces a large foodpellet, a fresh aliquot of the enriched sample is processed using the preclarificationprotocol. The sample pellet is lysed and processed on the MagMAX™ Express-96instrument. Then, Binding Solution and Magnetic Particles are added to the samples,and after washing by the MagMAX™ Express-96 instrument, nucleic acid is elutedwith 140 µL of Elution Buffer. The eluted DNA sample is ready for downstream PCR.

Centrifuge sample after enrichment

Centrifuge 1 mL of sample at 12, 000–16,000 × g for about 3 minutes.

q

If no large pellet: remove and discard the supernatant.

q

If a large pellet: perform the preclarification protocolon a fresh, 1-mL aliquot of the enriched sample.

q

Set up the Lysis Plate

Resuspend pellet in 300 μL of Lysis Buffer.

q

Transfer the sample to a MagMAX™ Express-96 Deep Well Plate (Lysis Plate).

q

Set up the MagMAX™ Express-96 processing plates

Tip Comb plate: Standard plate + Deep Well Tip Comb

Elution Plate: 140 µL Elution Buffer

Wash Plate 1: 300 µL Wash Buffer

Wash Plate 2: 300 µL Wash Buffer

q

Process samples on the MagMAX™ Express-96 instrument

Select program 44000799DWPrepSEQGN, select Start, and load processing plates into the instrument.

q

After 18 minutes, add 30 µL of Magnetic Particles and 180 µL of Binding Solution. Reload the plate, and pressStart.

q

When the MagMAX™ Express-96 run is complete, DNA is in Elution Buffer (Elution Plate).

q

Proceed to PCR, or seal the plate and store the DNA below –18°C

Workflow

Chapter 3 Isolate DNA with the PrepSEQ® Nucleic Acid Extraction KitProcedure overview3


Before first use of the kit

Before using a new PrepSEQ® Nucleic Acid Extraction Kit, prepare the reagents:• Binding Solution—Add approximately 35 mL of 100% isopropanol to an empty

Binding Solution bottle. Label the bottle to indicate that isopropanol is added.• Wash Buffer—Add 74 mL of 95% ethanol to the Wash Buffer Concentrate bottle,

and mix well. Label the bottle to indicate that ethanol is added.

Before each use of the kit

IMPORTANT! Mix the particles vigorously before each use, to ensure that all salts aredissolved.

White precipitate occasionally forms in the Magnetic Particles tube. Extractionexperiments show that formation of precipitate does not affect performance as long asthe precipitate is dissolved prior to use.

1. Incubate the tube of Magnetic Particles at 37±1°C for approximately 10 minutes.

2. Vortex for approximately 10 seconds.

Note: If the white precipitate is not completely dissolved after 10 minutes at37°C, apply longer incubation and higher temperatures (up to 50°C).

3. Keep at room temperature (23±5°C) until ready for use.

Centrifuge the enriched culture

IMPORTANT! Use proper aseptic technique while handling samples to avoid cross-contamination.

1. Transfer 1 mL of enriched culture to a 1.5-mL microcentrifuge tube.

2. Centrifuge the tube at 12,000–16,000 × g for about 3 minutes.(Optional) If the sample creates a large pellet, follow “For large sample pellets:perform the preclarification protocol” on page 14 with a fresh sample of the en-riched culture.

3. Remove and discard the supernatant as quickly as possible to prevent dissipationof pellet.(Optional) If necessary, follow “For high-fat samples: remove fat layer beforelysis” on page 14.

Prepare thereagents

ResuspendMagnetic Particles

Chapter 3 Isolate DNA with the PrepSEQ® Nucleic Acid Extraction KitBefore first use of the kit 3


For samples that produce a large pellet upon initial centrifugation (such as found withchocolate, dry pet food, and peanut butter samples), follow this preclarificationprotocol:

1. Transfer a fresh, 1 mL sample of enriched culture to a 1.5-mL microcentrifugetube.

2. Centrifuge the tube containing the sample at about 4000 × g for about 1 minute.

3. Transfer the supernatant to a new 1.5-mL microcentrifuge tube withoutdisturbing the pellet. Discard the pellet.

4. Centrifuge the tube containing the supernatant at 12,000–16,000 × g for about3 minutes.

5. Remove and discard the supernatant as quickly as possible to prevent dissipationof pellet.(Optional) If necessary, follow “For high-fat samples: remove fat layer beforelysis” on page 14.

For samples that contain a distinct, top, fat layer following centrifugation, remove thefat layer and supernatant as follows:

Type of fat layer Fat layer and supernatant removal

Liquid (for example,as found in milk orsoft cheese samples)

1. Use a P1000 pipettor to remove fat from the top surface byaspirating in a circular motion without disturbing the pellet.

2. Continue to collect supernatant from the top surface until allthe supernatant is removed.

3. Discard the supernatant into a waste container.

Solid fat layer (forexample, as found ininfant formulasamples)

1. Use a pipettor to gently dislodge the fat layer withoutdisturbing the pellet.

2. Aspirate the supernatant from the top surface using apipettor until all the supernatant is removed.

3. Discard the supernatant into a waste container.

Set up the Lysis Plate

1. Add 300 µL of the Lysis Buffer to the tube.

2. Resuspend by pipetting up and down.Alternatively, vortex until the pellet is resuspended, and quick spin for about5 seconds to remove the Lysis Buffer from the tube lid.

3. Transfer the sample to a MagMAX™ Express-96 Deep Well Plate (Lysis Plate).

4. (Optional but recommended) Set up a negative extraction control (NEC)containing 300 µL of Lysis Buffer in the Lysis Plate.

For large samplepellets: performthepreclarificationprotocol

For high-fatsamples: removefat layer beforelysis

Chapter 3 Isolate DNA with the PrepSEQ® Nucleic Acid Extraction KitSet up the Lysis Plate3


Set up the MagMAX™ Express-96 processing plates

Set up the MagMAX™ Express-96 processing plates as described in the followingtable.

Plate MagMAX™ Express-96plate type Action

Tip Comb Standard Place a 96-well Deep Well Tip Comb ina standard plate.

Elution Plate Standard Add 140 µL of Elution Buffer to eachsample and control well.

Wash Plate 1 Deep Well Add 300 μL of Wash Buffer to eachsample and control well.

Wash Plate 2 Deep Well Add 300 μL of Wash Buffer to eachsample and control well.

Process sample on MagMAX™ Express-96 Magnetic ParticleProcessor

1. Power on the MagMAX™ Express-96 Magnetic Particle Processor, select program44000799DWPrepSEQGN using the Up-arrow and Down-arrow keys, and pressStart.

2. Load the prepared plates according to the readout on the instrument, verifyingthat their orientation is {A1 to A1}.

Plate Action

Tip Comb Load the tip comb, then press Start.

Elution Plate Load the Elution Plate, then press Start.

Wash Plate 2 Load Wash Plate 2, then press Start.

Wash Plate 1 Load Wash Plate 1, then press Start.

Lysis Plate Load Lysis Plate, then press Start.

3. After 18 minutes, when prompted by the MagMAX™ Express-96 MagneticParticle Processor, dispense the Magnetic Particles and Binding Solution.

a. Vortex the Magnetic Particles for about 10 seconds until resuspension iscomplete.

b. Add 30 µL of Magnetic Particles to each well.

c. Add 180 µL of Binding Solution to each well.

d. Load the plate back into the instrument. Press Start.

4. When DNA sample preparation is complete (“Enjoy your DNA” is displayed onthe screen), remove the Elution Plate from the instrument.

Chapter 3 Isolate DNA with the PrepSEQ® Nucleic Acid Extraction KitSet up the MagMAX™ Express-96 processing plates 3


The Elution Plate contains DNA in Elution Buffer.

STOPPING POINT (Optional) If storage of the DNA sample before PCR is desired,seal the plate and store below –18°C.

5. Proceed to PCR (next chapter), using 30 µL of eluate for each assay.

Chapter 3 Isolate DNA with the PrepSEQ® Nucleic Acid Extraction KitProcess sample on MagMAX™ Express-96 Magnetic Particle Processor3


PCR with the MicroSEQ® Salmonellaspp. Detection Kit and RapidFinder™

Express Software

Product description

The MicroSEQ® Salmonella spp. Detection Kit detects Salmonella spp. simply, reliably,and rapidly in food samples using a lyophilized reagent format. The assay uses thepolymerase chain reaction (PCR) to amplify a unique microorganism-specific DNAtarget sequence and a TaqMan® probe to detect the amplified sequence.

The MicroSEQ® assay beads contain all the components necessary for the real-timePCR reaction: Salmonella spp.-specific probe and primers, enzyme, and other buffercomponents. The assay beads also contain an internal positive control (IPC) probe,primers, and template, to monitor for PCR inhibition. Pathogen Detection NegativeControl is included in the kit. Unknown samples and positive control samples areprovided by the investigator.

RapidFinder™ Express Software provides online, step-by-step instructions to set upthe real-time PCR assays followed by automated data analysis. Online help isprovided within the software. The software is designed for use on the AppliedBiosystems® 7500 Fast Real-Time PCR Instrument.

Kit contents and storage

Table 2 MicroSEQ® Salmonella spp. Detection Kit [96 reactions; Cat. no. 4403930]

Component Description Amount Cap color Storage

MicroSEQ® Salmonellaspp. Detection Kit

Salmonella spp. Assay Beads,8-tube strips

12 strips (96 tubes)

1 rack

Green (rack) 5±3°C

Protect fromlight andmoisture.[1]MicroAmp® Optical 8-Cap

Strips12 strips (96 caps) N/A

Pathogen DetectionNegative Control

Pathogen Detection NegativeControl

1.5 mL Red 5±3°C

[1] Excessive exposure to light may affect the fluorescent probes. To protect the beads from moisture, do not remove the desiccant from the pouch, and seal the pouch tightly each time you remove assay bead strips.

Note: Kit parts may ship separately, depending on the configuration ordered andstorage conditions.

4


Required materials not included with the kit

Unless otherwise indicated, all materials are available from Life Technologies. MLS:major laboratory supplier.

Item Source

Instrumentation and equipment

Applied Biosystems® 7500 Fast Real-Time PCRInstrument

Contact your local LifeTechnologies representative.

7500 Fast Precision Plate Holder for MicroAmp®

Tube StripsCat. no. 4403809

MicroAmp® 96-Well Base Cat. no. N8010531

MicroAmp® Cap Installing Tool Cat. no. 4330015

MicroAmp® Multi-removal Tool Cat. no. 4313950

Benchtop microcentrifuge with 8-tube strip adapter MLS

(Optional but recommended) Plate centrifuge MLS

Block heater MLS

Laboratory mixer (Vortex mixer or equivalent) MLS

Pipettors:

• Positive-displacement

• Air-displacement

• Multichannel

MLS

Consumables

Aerosol-resistant pipette tips MLS

Disposable gloves MLS

MicroAmp® Fast 8-Tube Strip, 0.1-mL Cat. no. 4358293

MicroAmp® Optical 8-Cap Strip, 300 strips Cat. no. 4323032

Reagents

Nuclease-free Water Cat. no. AM9938

Chapter 4 PCR with the MicroSEQ® Salmonella spp. Detection Kit andRapidFinder™ Express SoftwareRequired materials not included with the kit

4


Workflow

Create or edit a run file in RapidFinder™ Express Software

q

Prepare the assay beads

q

Set up the PCR reactions

q

Load and run the reactions

q

View results and data analysis

Important procedural guidelines

IMPORTANT! Seal the tubes with the transparent, optical strip-caps provided in thekit. Always use intact 8-cap strips, even if empty tubes have been added next toreaction tubes.

Do not use colored caps or tubes for real-time PCR reactions. Colored caps or tubesmay affect dye-signal readings during real-time PCR.

• RapidFinder™ Express Software determines the plate layout and therefore mustbe set up before distributing DNA samples for the real-time PCR.

• Cut the storage pouch above the zip-lock strip so that it can be resealed.• Follow these additional tips when setting up the PCR:

– 8-tube strips can be cut apart with scissors.If necessary, trim any remaining connector material from the cut to allow abetter fit against adjacent tubes in the 7500 Fast Precision Plate Holder forMicroAmp® Tube Strips.

– Use a new pipette tip for each sample.– If you mix the assay beads with the DNA samples by pipetting up and

down, keep the pipette tip at the bottom of the tube to minimize aerosolformation and cross-contamination.

– Follow the recommendations in “Good laboratory practices for PCR/RT-PCR” on page 30.

Chapter 4 PCR with the MicroSEQ® Salmonella spp. Detection Kit andRapidFinder™ Express Software

Workflow

4


If you see this in theElution Plate... Do this...

Oil droplets as a toplayer

After lysis, food samples with high fat or oil content can form a toplayer containing fat and debris over the aqueous phase containingthe DNA. Collect the DNA sample for PCR from the clear middlephase, avoiding the top fatty layer and bottom pellet (Figure 1).

Magnetic Particles 1. Place the Elution Plate on a 96-well magnetic ring stand forat least 1 minute.

2. Collect eluate for PCR, while the Elution Plate remains onthe magnetic stand.Avoid touching the Magnetic Particles.

Particulate residuefrom food sample

If the particulate residue is not removed using a 96-well magneticring stand:

1. Centrifuge the Elution Plates at about 4000 × g for about30 seconds.

2. Avoid the particulate residue, and collect eluate for PCR.

Avoid top layer containing fat/oil

Collect sample for PCR from below fat/oil layer

Avoid particulate material from column

Figure 1 High-fat samples: Collect sample from middle phase after lysis.

Create or edit a run file in RapidFinder™ Express Software

On the main page of the RapidFinder™ Express Software, select Create/Edit a RunFile , and enter the target pathogen, number of samples, replicates, and positiveand negative controls for each target at the prompts.The software determines the sample layout based on the information entered, andcreates a run file.

Prepare the assay beads

Follow the plate layout determined by the RapidFinder™ Express Software. (InRapidFinder™ Express Software v1.1, select Pipette Samples for step-by-step in-structions.)

1. Transfer the appropriate number of individual tubes or 8-tube strips from thestorage pouch to a 96-well base at room temperature (23±5°C), one tube for eachreaction.

Avoid fat layer andparticulates aftersample lysis(collection of DNAsample for PCR)

Chapter 4 PCR with the MicroSEQ® Salmonella spp. Detection Kit andRapidFinder™ Express SoftwareCreate or edit a run file in RapidFinder™ Express Software

4


2. Seal the storage pouch using the zip-lock strip, and store the pouch at 5±3°C.

IMPORTANT! Do not remove the desiccant from the storage pouch.

3. If required by the software-determined layout, place empty MicroAmp® Fast8-Tube Strips (or partial strips) to balance the tray when the assay tubes areplaced in the instrument later.


In the RapidFinder™ Express Software, select Pipette Samples on the main page,select the appropriate run file, and follow the software prompts in the followingprocedure.

1. If necessary, thaw samples and controls completely, and mix each sample orcontrol thoroughly.If the Elution Plate contains oil droplets, magnetic particles, or food particulateresidue, see “Avoid fat layer and particulates after sample lysis (collection ofDNA sample for PCR)” on page 20.

2. To avoid cross-contamination, we recommend a brief centrifugation of allsamples and controls to bring the contents to the bottom of the wells or tubes.

3. Following the layout determined by RapidFinder™ Express Software, add 30 µLof sample or control to each assay bead at room temperature (23±5°C), and mixby gently pipetting up and down a few times.Beads dissolve in 1–5 seconds.Alternatively, vortex the assay tubes after they are capped in the final step.

4. Seal the tubes with the transparent, optical strip caps provided in the kit.Use the MicroAmp® 96-Well Base and the MicroAmp® Cap Installing Tool toavoid collapsing, bending, or misaligning the tubes. Confirm that the strips arestraight and that each tube is in line with the adjacent tube.

5. Mark or label one end of the strip cap (but not directly over any one cap) tomaintain the strip orientation when transferring the tubes to the instrument tray.

6. Make sure that the reactions are thoroughly mixed: if reactions were notpreviously mixed during the pipetting step, place the assay tubes in a 96-wellbase and vortex to mix.

7. Make sure that the reagents are at the bottom of tubes: briefly centrifuge the striptubes at 200–600 × g for about 20 seconds using a centrifuge with a plate adapteror a benchtop microcentrifuge with an 8-strip PCR tube adapter.



4


Load and run the reactions

In the RapidFinder™ Express Software, select Start Instrument Run on the mainpage, select the appropriate run file, and follow the software prompts in the followingprocedure.

1. To load the instrument, transfer the tubes to the 7500 Fast instrument in the sameconfiguration as the run layout.Use the 7500 Fast Precision Plate Holder for MicroAmp® Tube Strips in theinstrument.

2. Close the tray to the instrument.

3. Follow the RapidFinder™ Express Software prompts to start the run.

Figure 2 Transfer tubes to the 7500 Fast instrumentRapidFinder™ Express Software directs the user to load empty strip tubes in column 1 (far left)and column 12 (far right), if needed. The empty capped 8-tube strips evenly distribute theclamping load applied to the sample tube strips during processing, thereby minimizing the riskof collapsing any tubes.


In the RapidFinder™ Express Software, select View Results on the main page,select the appropriate run file, and follow the prompts to view results.Data analysis is automated by the software.

Chapter 4 PCR with the MicroSEQ® Salmonella spp. Detection Kit andRapidFinder™ Express SoftwareLoad and run the reactions

4


Follow the RapidFinder™ Express Software prompts for "Investigating WarningResults or Failed Runs in the SDS Software."

IMPORTANT! If you modify a RapidFinder™ Express Software run file in the SDSSoftware, you cannot open the run file again in the RapidFinder™ Express Software.To avoid altering a RapidFinder™ Express Software run file, save the run file under anew name in the SDS software before performing any actions.

1. From View Results in the RapidFinder™ Express Software, select and open therun file, and then click View in SDS.

2. Select File4Save As, and save the run file under a new name.

If necessary,investigate resultsin SDS Software



4


Recommended confirmationmethods

In terms of AOAC® validation, enriched cultures with positive PCR results weretested further by cultural confirmation using the appropriate reference method for thesample matrix (see “AOAC Performance Tested Methods Certification” on page 30).

5


Troubleshooting

Observation Possible cause Recommended action

Difficult to avoid bacterial pelletduring removal of supernatant

The sample was leftunattended before aspiratingoff the supernatant, causingdissipation of the bacterialpellet.

Remove the supernatant immediately followingcentrifugation.

The size of the bacterial pelletis very small and difficult tosee.

Remove the supernatant carefully, leavingbehind up to 50 µL of supernatant, to avoidaspiration.

Difficult to resuspend bacterialpellet during lysis

Pellet is too hard. Ensure maximum resuspension of the pellet inthe Lysis Buffer or Proteinase K Buffer beforeproceeding.

Transfer the entire contents, including theincompletely resuspended pellet (if any) to theLysis Plate.

Follow the PrepSEQ® Preclarification Protocol(“For large sample pellets: perform thepreclarification protocol” on page 14).

Inhibition of downstream PCR,indicated by nondetection ofIPC reaction

Magnetic Particles were in theElution Plate.

Avoid disturbing the Magnetic Particles duringtransfer of eluted DNA to the lyophilized assay.

Avoid transfer of Magnetic Particles using oneof the following methods (optional):

• Place the Elution Plate on the 96-WellMagnetic Ring Stand during transfer ofeluted DNA sample to the lyophilizedassay.

• Spin the plate at maximum speed in aplate centrifuge for the equivalent ofapproximately 4,000 × g for approximately30 seconds, to pellet the MagneticParticles to the bottom of the plate.

Elution Plate containsincompletely removedparticulate residue from thefood sample.

Avoid residue during transfer of eluted DNA tothe lyophilized assay.

(Optional) Spin the plate at maximum speed ina plate centrifuge for the equivalent ofapproximately 4,000 × g for approximately30 seconds, to pellet the food residue to thebottom of the plate.

Removal of samplesupernatant before addition oflysis buffer was incomplete.

Ensure maximal removal of the supernatantwithout disturbing the bacterial pellet.

A



In positive control wells, no IPCsignal is detected, but target-specific signal is detected.

A high copy number of targetDNA exists in samples, result-ing in preferential amplificationof the target-specific DNA.

No action is required. The result is consideredpositive.

In positive control wells, no tar-get-specific signal is detected.

Positive control was omitted(pipetting error).

Repeat the assay. Make sure to pipet thepositive control into all positive-control wells.

In negative control wells, noIPC signal is detected, but atarget-specific signal is detec-ted.

Carryover contaminationcaused target signal in negativecontrol wells.

Additionally, no IPC signal innegative control wells can becaused by:

• A high copy number oftarget DNA exists insamples, resulting inpreferential amplificationof the target-specific DNA.

• A problem occurred withIPC amplification.

To correct carryover contamination, repeat theassay using fresh aliquots of all reagents andclean pipetting equipment.

To determine whether IPC amplification is aproblem, examine unknown wells for an IPCsignal. If an IPC signal is present, IPCamplification is not a problem.

In negative control wells, tar-get-specific signal is detected.

Carryover contaminationoccurred.

1. Repeat the assay using fresh aliquots ofall reagents and clean pipettingequipment.

2. If the negative control continues to showcontamination, repeat the assay using anew kit.

3. If the negative control continues to showcontamination, contact Life TechnologiesTechnical Support.

In unknown wells, no IPC ortarget-specific signal is detec-ted.

Inhibition of PCR occurred. Dilute the sample 1:5 with Nuclease-freeWater, to dilute PCR inhibitors, and repeat theassay. If PCR remains inhibited, repeat thesample preparation.

Refer to other troubleshooting suggestions forremoval of Magnetic Particles or particulateresidue from the DNA sample.

In unknown wells, no IPC signalis detected, but target-specificsignal is detected.

A high copy number of targetDNA exists in samples,resulting in preferentialamplification of the target-specific DNA.

No action is required. The result is consideredpositive.

Appendix A TroubleshootingA



Multicomponent plot signalsfor FAM™, VIC®, and ROX™ de-tectors increase/decreasesduring cycles 1–15, but the am-plification curve and result arenot affected (this observationapplies to View in SDS mode).

Incomplete mixing and dissolu-tion of the lyophilized bead withsample or control.

After addition of 30 µL of sample or PathogenNegative Control to the bead and capping thetubes:

1. Vortex strips at high speed for about10 seconds, and centrifuge the strips at200–600 × g for about 10 seconds.

2. Vortex the strips again on high speed forabout 10 seconds, and centrifuge thestrips at 200–600 × g for about 1 minute.

Ensure that all liquid is at the bottom of thetubes and the beads are fully dissolved beforeproceeding.

Replicate results for a sampleare inconsistent.

All replicate wells for a sampledo not have the same result.

If more than two replicates yield the sameresult (for example, 2 of 3 replicates arenegative, but 1 replicate is positive), refer toyour laboratory protocol to determine whetherto repeat the assay using fresh samples andreagents.

If only 2 replicates were run and the resultsare not consistent, repeat the assay usingfresh samples and reagents.

Appendix A Troubleshooting A



Amplicon contamination. • Contamination wasintroduced into the PCRclean area from post-amplification reactiontubes that were eitheropened in the clean areaor brought into the PCRclean area fromcontaminated gloves orsolutions.

• Contamination wasintroduced into the real-time PCR instrument fromcrushed and broken PCRreaction tubes.

Prepare negative control samples using atleast one 8-tube strip of MicroSEQ® AssayBeads.

1. Divide the assay beads into two sets.

a. To the first set of assay beads, add30 μL of Nuclease-free Water.

b. To the second set of assay beads,add 29 μL of Nuclease-free Waterplus 1 μL of 1 U/μL Uracil DNAGlycosylase (Cat. no. 18054-015).

2. Run samples on the 7500 Fast Real-TimePCR Instrument using SDS software andselect Fast 7500 run mode.

3. Under the instrument tab:

• Select Add Step to stage 1 of the PCRcycle that consists of 10 minutes at50°C.

• Extend the 95°C step from20 seconds to 10 minutes.

Amplicon contamination is indicated by target-specific signal in the –UNG samples and notarget-specific signal in +UNG samples.

If the instrument block was contaminated,consult the 7300/7500/7500 Fast Real-TimePCR System Absolute Quantitation UsingStandard Curve Getting Started Guide (Pub.no. 4347825) and/or contact a servicerepresentative to clean the instrument.

Appendix A TroubleshootingA


Supplemental information

Product information

The sensitivity of the assay in real culture samples depends on the quality of thesample preparation method that is used. The AOAC®Performance Tested MethodsSM

workflow described in this user guide allows you to detect 1 to 3 colony-formingunits (CFU) in 25 grams of food or environmental swab and sponge samples.Following the workflow described in this user guide, the limit of detection is 103

cfu/mL (not part of AOAC® validation study).

The MicroSEQ® Salmonella spp. Detection Kit can detect all Salmonella enterica speciestested and did not detect any non-Salmonella species tested. The method does notallow detection of Salmonella bongori.

The MagMAX™ Express-96 Magnetic Particle Processor is for indoor use only.

Condition Acceptable range

Temperature 10–40°C

Humidity Maximum relative humidity 80% for temperatures up to 31°Cdecreasing linearly to 50% relative humidity at 40°C

The Applied Biosystems® 7500 Fast Real-Time PCR Instrument is for indoor use onlyand for altitudes not exceeding 2,000 m (6,500 ft) above sea level.

Condition Acceptable range

Temperature 15–30°C

Maximum change of less than 15°C per 24 hours

Humidity 20–80% relative humidity, noncondensing

B

Kit sensitivity

Kit specificity

Operationalconditions


AOAC® Performance Tested MethodsSM Certification

Visit www.lifetechnologies.com/foodsafety for a list of workflows for detection ofSalmonella spp.

Table 3 Performance Tested MethodsSM Certification of the workflow

Certification

The detection of Salmonella spp. using PrepSEQ® kits and MicroSEQ® Salmonella spp.Detection Kit has earned the Performance Tested MethodsSM Certification from theAOAC® Research Institute. The validated workflow described in this user guideincludes:

• Enrichment media: Buffered Peptone Water (with skim milk powder and BrilliantGreen Dye Solution for chocolate matrices)

• PrepSEQ® Nucleic Acid Extraction Kit• MicroSEQ® Salmonella spp. Detection Kit• Applied Biosystems® 7500 Fast Real-Time PCR Instrument• RapidFinder™ Express Software• Confirmation testing of positive samples.

Reference method Matrix

ISO 6579:2002 Food: raw ground beef, raw chicken wings, raw shrimp,cantaloupe, brie, dry infant formula, chocolate, dry petfood, shell eggs, black pepper

USFDA BAM, Chapter 5 Food: peanut butter

Environmental samples: stainless steel, sealed concrete,plastic, ceramic tile, rubber

Good laboratory practices for PCR/RT-PCR

When preparing samples for PCR/RT-PCR amplification:• Wear clean gloves and a clean lab coat (not previously worn while handling

amplified PCR/RT-PCR products or used during sample preparation).• Change gloves whenever you suspect that they are contaminated.• Maintain separate areas and dedicated equipment and supplies for:

– Sample preparation and PCR/RT-PCR setup.– PCR/RT-PCR amplification and analysis of PCR/RT-PCR products.

• Do not bring amplified PCR/RT-PCR products into the PCR/RT-PCR setup area.

Appendix B Supplemental informationAOAC® Performance Tested MethodsSM CertificationB



• Open and close all sample tubes carefully. Avoid splashing or spraying PCR/RT-PCR samples.

• Keep reactions and components capped as much as possible.• Use a positive-displacement pipettor or aerosol-resistant barrier pipette tips.• Clean lab benches and equipment periodically with 10% bleach solution or

DNAZap™ Solutions (Cat. no. AM9890).

Appendix B Supplemental informationGood laboratory practices for PCR/RT-PCR B


Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specifiedin the user documentation may result in personal injury or damage to theinstrument or device. Ensure that anyone using this product has receivedinstructions in general safety practices for laboratories and the safetyinformation provided in this document.

· Before using an instrument or device, read and understand the safetyinformation provided in the user documentation provided by themanufacturer of the instrument or device.

· Before handling chemicals, read and understand all applicable Safety DataSheets (SDSs) and use appropriate personal protective equipment (gloves,gowns, eye protection, etc). To obtain SDSs, see the “Documentation andSupport” section in this document.

C


Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,ensure laboratory personnel read and practice the general safety guidelines forchemical usage, storage, and waste provided below, and consult the relevantSDS for specific precautions and instructions:· Read and understand the Safety Data Sheets (SDSs) provided by the

chemical manufacturer before you store, handle, or work with any chemicalsor hazardous materials. To obtain SDSs, see the “Documentation andSupport” section in this document.

· Minimize contact with chemicals. Wear appropriate personal protectiveequipment when handling chemicals (for example, safety glasses, gloves, orprotective clothing).

· Minimize the inhalation of chemicals. Do not leave chemical containers open.Use only with adequate ventilation (for example, fume hood).

· Check regularly for chemical leaks or spills. If a leak or spill occurs, followthe manufacturer's cleanup procedures as recommended in the SDS.

· Handle chemical wastes in a fume hood.· Ensure use of primary and secondary waste containers. (A primary waste

container holds the immediate waste. A secondary container contains spillsor leaks from the primary container. Both containers must be compatiblewith the waste material and meet federal, state, and local requirements forcontainer storage.)

· After emptying a waste container, seal it with the cap provided.· Characterize (by analysis if necessary) the waste generated by the particular

applications, reagents, and substrates used in your laboratory.· Ensure that the waste is stored, transferred, transported, and disposed of

according to all local, state/provincial, and/or national regulations.· IMPORTANT! Radioactive or biohazardous materials may require special

handling, and disposal limitations may apply.

CAS Chemical information

26628-22-8 Sodium Azide Sodium azide may react with lead and copperplumbing to form highly explosive metal azides.

50-01-1 Guanidine HCl Contact with acids or bleach liberates toxic gases.DO NOT ADD acids or bleach to any liquid wastescontaining this product.

593-84-0 GuanidineIsothiocyanate

Contact with acids or bleach liberates toxic gases.DO NOT ADD acids or bleach to any liquid wastescontaining this product.

Specific chemicalhandling

Appendix C SafetyChemical safety C


Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,infectious agents, and blood of humans and other animals have the potential totransmit infectious diseases. All work should be conducted in properlyequipped facilities using the appropriate safety equipment (for example,physical containment devices). Safety equipment also may include items forpersonal protection, such as gloves, coats, gowns, shoe covers, boots,respirators, face shields, safety glasses, or goggles. Individuals should betrained according to applicable regulatory and company/ institutionrequirements before working with potentially biohazardous materials. Followall applicable local, state/provincial, and/or national regulations. The followingreferences provide general guidelines when handling biological samples inlaboratory environment.

· U.S. Department of Health and Human Services, Biosafety in Microbiologicaland Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)21-1112, Revised December 2009; found at:www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Appendix C SafetyBiological hazard safetyC


http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Documentation and support

Obtaining SDSs

Safety Data Sheets (SDSs) are available from www.lifetechnologies.com/support.

Note: For the SDSs of chemicals not distributed by Life Technologies Corporation,contact the chemical manufacturer.

Obtaining Certificates of Analysis

The Certificate of Analysis provides detailed quality control and product qualificationinformation for each product. Certificates of Analysis are available on our website. Goto www.lifetechnologies.com/support and search for the Certificate of Analysis byproduct lot number, which is printed on the box.

Obtaining support

For the latest services and support information for all locations, go to:

www.lifetechnologies.com/support

At the website, you can:• Access worldwide telephone and fax numbers to contact Technical Support and

Sales facilities• Search through frequently asked questions (FAQs)• Submit a question directly to Technical Support ([email protected])• Search for user documents, SDSs, vector maps and sequences, application notes,

formulations, handbooks, certificates of analysis, citations, and other productsupport documents

• Obtain information about customer training• Download software updates and patches

Food Safety support

Website: www.lifetechnologies.com/foodsafety

Support email: [email protected]

Phone number in North America: 1-800-500-6855


http://www.lifetechnologies.com/support



mailto:[email protected]



Phone number outside of North America: Visit www.lifetechnologies.com/support,select the link for phone support, and select the appropriate country from thedropdown menu.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forthin the Life Technologies' General Terms and Conditions of Sale found on LifeTechnologies' website at www.lifetechnologies.com/termsandconditions. If you haveany questions, please contact Life Technologies at www.lifetechnologies.com/support.

Documentation and SupportLimited product warranty



http://www.lifetechnologies.com/termsandconditions



References

ISO. 2002. Microbiology of food and animal feeding stuffs – Horizontal method forthe detection of Salmonella spp. Reference number 6579:2002.

U.S. Food and Drug Administration, Bacteriological Analytical Manual (BAM),Chapter 5; go to www.fda.gov/food/foodscienceresearch/laboratory methods/ucm2006949.htm and scroll to Salmonella. Accessed 23 Sept. 2013.


http://www.fda.gov/food/foodscienceresearch/laboratory methods/ucm2006949.htm

http://www.fda.gov/food/foodscienceresearch/laboratory methods/ucm2006949.htm

Headquarters5791 Van Allen Way | Carlsbad, CA 92008 USA | Phone +1 760 603 7200 | Toll Free in USA 800 955 6288For support visit lifetechnologies.com/support or email [email protected]

lifetechnologies.com

8 November 2013

http://lifetechnologies.com/support


http://lifetechnologies.com

Documents

Real-time PCR Detection of Salmonella spp. in Food and · PDF file · 2013-12-13For testing of Food and Environmental samples only. ... MagMAX™ Express-96 Magnetic Particle Processor