USER GUIDE
Real-Time PCR Detection of Salmonella spp. in
Primary Production Samples
Automated or spin-column DNA isolation
for use with:
PrepSEQ® Nucleic Acid Extraction Kit
PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean with Proteinase K
MicroSEQ® Salmonella spp. Detection Kit
Publication Number 4476867
Revision B
ABI 29/02 – 09/10
ALTERNATIVE ANALYTICAL METHODS
FOR AGRIBUSINESS
www.afnor-validation.com
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 OR IMPLIED,
INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE 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 CONSEQUENTIAL DAMAGES 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 the product
(a) to perform internal research for the sole benefit of the purchaser; and (b) for environmental testing, quality control/quality assurance testing, food and
agricultural testing, including reporting results of purchaser's activities in environmental testing, quality control/quality assurance testing, food and
agricultural testing for a fee or other commercial consideration. No other right is hereby granted expressly, by implication, or by estoppel. This product is for
environmental testing, quality control/ quality assurance testing, food and agricultural testing and research purposes only.
The purchase of this product does not grant the purchaser any additional rights, including (without limitation) the right to transfer or resell the product in
any form or the right to use the product as a therapeutic agent or diagnostics test component. For information on obtaining additional rights, please contact
[email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.
TRADEMARKS
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. NF Validation is a registered trademark of
Association Francaise de Normalisation (AFNOR). Stomacher is a registered trademark of Seward Limited. Whirl-Pak is a registered trademark of Aristotle
Corporation.
© 2014 Thermo Fisher Scientific Inc. All rights reserved.
Contents
About this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
■ CHAPTER 1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Intended user . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Shelf life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
NF Validation™ by AFNOR certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Expiration date . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Operational conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Altitude . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Definitions of terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
■ CHAPTER 2
PrepSEQ® Nucleic Acid Extraction Kit: Salmonella spp. 11
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Kit contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Materials not included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Pre-enrichment of primary production stage samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Homogenization and primary enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Secondary enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Automated sample preparation using PrepSEQ® Nucleic Acid Extraction Kit . . . . . . . . . . . . . . . . . . 13
Prepare PrepSEQ® kit materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Prepare the MagMAX™ Express-96 processing plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Run samples on the MagMAX™ Express-96 instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
■ CHAPTER 3 PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean
with Proteinase K: Salmonella spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Kit contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Materials not included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Pre-enrichment of primary production stage samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
3
Contents
Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Homogenization and primary enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Secondary enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Sample preparation using PrepSEQ Rapid Spin Sample Preparation Kit – Extra Clean with
Proteinase K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
■ CHAPTER 4
MicroSEQ® Salmonella spp. Detection Kit . . . . . . . . . . . 23
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Kit contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Materials not included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Prepare for PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Prepare PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Prepare tubes for the 7500 Fast, StepOne™, and StepOnePlus™ Systems . . . . . . . . . . . . . . . . . 28
Run the reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Run the 8-tube reactions on the 7500 Fast System (only) without RapidFinder™ Express
Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
View results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
General process without RapidFinder™ Express Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Resources for viewing results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Confirmation of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30
30
30
30
31
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
■ APPENDIX A
StepOne™ and StepOnePlus™ Systems . . . . . . . . . . . . . . 33
Run the 8-tube reactions on the StepOne™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Run the 8-tube reactions on the StepOnePlus™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
■ APPENDIX B
Good PCR practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
PCR good laboratory practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Plate layout suggestions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
■ APPENDIX C
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Obtaining Certificates of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
Contents
Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Food safety support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
5
About this guide
Revision history
Revision
Date
Description
B
September 2014
• Updated NF Validation™ certification wording to most recent requirements.
• Updated template with associated updates to the limited use label license
information, warranty information, trademark statement, and safety
statements.
A
6
November 2012
New user guide.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
1
Overview
IMPORTANT! Before using the products described in this guide, read and understand
the information in the “Safety” appendix in this document.
Description
We have developed a sample preparation workflow for detection of Salmonella spp. in
primary production samples. The workflow outlines enrichment, sample preparation,
and detection by real-time PCR. The following sample preparation methods are
available for use in detection of Salmonella spp. in primary production samples postenrichment:
• Magnetic bead-based method — see “PrepSEQ® Nucleic Acid Extraction Kit:
Salmonella spp.” on page 11
• Spin column-based method — see “PrepSEQ® Rapid Spin Sample Preparation
Kit – Extra Clean with Proteinase K: Salmonella spp.” on page 17
Both methods utilize a shared two-step enrichment prior to sample purification.
Visit www.lifetechnologies.com/foodsafety for a complete list of workflows for
detection of Salmonella spp. (Pub. no. MAN0009417).
Intended user
The intended users of the MicroSEQ® Salmonella spp. Detection Kit are microbiological
analysts who need to test for Salmonella spp. in food and environmental samples. The
MicroSEQ® Salmonella spp. Detection Kit is not for any animal or human therapeutic or
diagnostic use.
Shelf life
See expiration date as noted on the outer box label.
Specificity
The MicroSEQ® Salmonella spp. Detection Kit can detect all Salmonella enterica species
tested and did not detect any non-Salmonella species tested. The genus Salmonella
consists of the two species Salmonella enterica and Salmonella bongori. Salmonella enterica
incorporates the most important clinical serovars for humans. The method does not
allow detection of Salmonella bongori.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
7
1
Chapter 1 Overview
NF Validation™ by AFNOR certification
NF Validation™ by AFNOR certification
ABI 29/02 – 09/10
ALTERNATIVE ANALYTICAL
METHODS FOR AGRIBUSINESS
www.afnor-validation.com
The PrepSEQ® Nucleic Acid Extraction Kit, the PrepSEQ® Rapid Spin Sample
Preparation Kit – Extra Clean with Proteinase K, and MicroSEQ® Salmonella spp.
Detection Kit are part of the NF Validation™ workflow to recover Salmonella spp. DNA
from broth cultures of environmental samples commonly found in the primary
production stage. The certification uses the ISO 16140 standard for the validation of
alternative methods (Alternative Analytical Methods for Agribusiness. Certified by NF
Validation™; www.afnor-validation.com). This kit was compared and found
equivalent to the ISO 6579 reference method. The validated workflow includes:
• One of the following sample preparation kits:
– The PrepSEQ® Nucleic Acid Extraction Kit
– The PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean with
Proteinase K
• The MicroSEQ® Salmonella spp. Detection Kit
• The Applied Biosystems® 7500 Fast Real-Time PCR Instrument
• RapidFinder™ Express Software
Reference method
Matrix
Two reference methods were used during the
validation study:
Environmental samples: boot socks
(poultry and pigs), feces (poultry and
pigs), litter, animal drinking water, and
sponges
• NF EN ISO 6579:2002/Amd 1:2007:
Microbiology of food and animal feeding stuffs.
Horizontal method for the detection of
Salmonella spp. Detection of Salmonella spp. in
animal faeces and in environmental samples
from the primary production stage
• NF U47-100: French norm on isolation and
identification of Salmonella spp. in the
environment and samples from animal
productions: "Recherche par l’isolement et
l’identification de tout sérovar ou de sérovar(s)
spécifié(s) de salmonelles dans
l’environnement des productions animales
(Detection by streaking of any Salmonella
serotypes in primary production samples)
General remarks and recommendations:
• Comply with Good Laboratory Practices — GLP; refer to EN ISO 7218 standard.
• We recommend that ISO 6579 and ISO 6887 standards be followed for the
preparation of “master suspensions”.
• In the context of NF validation™ certification, samples of more than 25 grams
have not been tested.
Expiration date
8
For more information about the expiration date of the NF Validation™ certification,
please refer to the certificate, ABI 29/02 – 09/10, available at www.afnorvalidation.com or in the Product Literature section on the product page at
www.lifetechnologies.com.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
Chapter 1 Overview
Operational conditions
1
Operational conditions
Altitude
The Applied Biosystems® 7500 Fast Real-Time PCR System is for indoor use only and
for altitudes not exceeding 2,000 m (6,500 ft.) above sea level.
Temperature and humidity requirements
Condition
Acceptable range
Temperature
15 to 30°C (50 to 90°F)
Maximum change of less than 15°C (59°F) per 24 hours
Humidity
20 to 80% relative humidity, noncondensing
Definitions of terms
This protocol uses the following terms:
• Amplification – The process of making copies of, and thereby increasing the
amount of, a specific DNA sequence.
• Internal Positive Control (IPC) – A control in all reaction wells that should
always yield amplification. If it does not, a problem with amplification exists. The
presence of an inhibitor in the unknown sample is indicated if the IPC signal is
significantly reduced.
• Negative control – A reaction mixture that lacks a target sequence. It indicates
contamination if amplification occurs, or an amplification problem if the IPC
signal is reduced or absent. The Pathogen Detection Negative Control is provided
in the kit as a negative control. At least one negative control is required for each
target assay.
• Polymerase Chain Reaction (PCR) – Technology used to amplify, or increase the
amount of a DNA sequence.
• Positive control – A control that establishes the expected amplification of a target.
The lack of a target signal in a positive control well indicates a pipetting error or a
problem with amplification. A positive control is provided by the investigator
and is recommended but not required for each run.
• Primer – A segment of DNA that is complementary to the target DNA sequence
or IPC DNA sequence. It is needed to start amplification.
• Probe – A segment of DNA that is complementary to the target DNA sequence or
IPC DNA sequence. The probe is labeled with a reporter dye. When the probe
binds to the target or IPC during the amplification step, fluorescence is emitted.
The Sequence Detection System (SDS) or Real-Time PCR System detects the
fluorescence, indicating the presence of the target or IPC DNA sequence.
• Target – The bacteria being tested.
• Unknown sample – A DNA sample from a food substance that you test for the
presence of one or more food pathogens.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
9
1
Chapter 1 Overview
Workflow
Workflow
A sample preparation workflow is shown below for both the magnetic bead-based
(see page 11) as well as the spin-column based method (see page 17).
Combine sample with TT Broth at a 1:9 ratio (e.g. 25 g or 25 mL sample:25 mL TT Broth)
Enrich samples at 37±1°C for 16–20 hr
Dilute enriched sample with BPW at a 1:9 ratio (1 mL of sample:9 mL of BPW)
Enrich samples at 37±1°C for 4–6 hr
PrepSEQ® Nucleic Acid Extraction Kit
automated on MagMAX™ Express-96
PrepSEQ® Rapid Spin Kit –
Extra Clean with Proteinase K
Real-time PCR: use the MicroSEQ® Salmonella spp. Detection Kit
with the 7500 Fast Instrument
10
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
2
PrepSEQ® Nucleic Acid Extraction
Kit: Salmonella spp.
Overview
The PrepSEQ® Nucleic Acid Extraction Kit provides a simple way to prepare DNA
from primary production stage environmental samples. This kit is validated for use
with primary production stage environmental samples. The procedure involves:
• Salmonella spp. enrichment; primary enrichment in Tetrathionate Broth (TT Broth)
followed by dilution and secondary enrichment in Buffered Peptone Water (BPW)
• Automated sample extraction via the MagMAX™ Express-96 Magnetic Particle
Processor
The recommended starting samples are supported:
• Boot socks and sponges: 100 to 150 mL of TT Broth
• Swabs: 10 mL of TT Broth
• Other environmental samples (such as feces): TT Broth at a 1:10 ratio; for example,
25 g of sample with 225 ml of TT Broth
Kit contents
PrepSEQ® Nucleic Acid Extraction Kit
Cat. nos. 4480466 (100 rxns), 4428176 (300 rxns‡)
Item
Quantity or volume
Storage
Lysis Buffer
2 bottles, 50 mL/bottle
Room temperature (23±5°C)
Magnetic Particles
2 tubes, 1.5 mL/tube
5±3°C
Binding Solution (Isopropanol)
1 empty bottle
Room temperature (23±5°C)
Wash Buffer Concentrate
2 bottles, 26 mL/bottle
Room temperature (23±5°C)
Elution Buffer
1 bottle, 25 mL
Room temperature (23±5°C)
Proteinase K (PK) Buffer
1 bottle, 50 mL
Room temperature (23±5°C)
Proteinase K (20 mg/mL)
1 tube, 1.25 mL
Below –18°C
‡ The 300 reaction kit (Cat. no. 4428176) contains 3 times the quantity/volume shown in table.
Note: Parts may ship separately depending on the configuration ordered and storage
conditions.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
11
2
Chapter 2 PrepSEQ® Nucleic Acid Extraction Kit: Salmonella spp.
Materials not included
Materials not included
The following table includes materials and equipment for using (but not included in)
the PrepSEQ® Nucleic Acid Extraction Kit for Food Testing. Unless otherwise
indicated, all materials are available from Life Technologies. MLS: Major laboratory
supplier.
Equipment, consumables, and reagents
Item
Source
Equipment
96-Well Magnetic Ring Stand
Cat. no. AM10050
Block heater or water bath (37–50°C)
MLS
Plate centrifuge
Eppendorf 5804 or equivalent
MagMAX™ Express-96 Deep Well Magnetic
Particle Processor
Cat. no. 4400079
Homogenizer (Stomacher® 400 Laboratory
Blender)
Seward Cat. no. 0400/001/AJ or
equivalent
Vortexer
MLS
Consumables
Disposable gloves
MLS
Micropipette tips, aerosol-resistant
MLS
Pipettors:
MLS
• Positive-displacement
• Air-displacement
• Multichannel
MagMAX™ Express-96 Deep Well Tip Combs
Cat. no. 4388487
MagMAX™ Express-96 Deep Well Plates
Cat. no. 4388476
MagMAX™ Express-96 Standard Plates
Cat. no. 4388475
Whirl-Pak® Filter Bags, 6” x 9”, 24 oz., 250/pkg
(Stomacher® bags with mesh)
Nasco Cat. no. BO1348WA or
equivalent
Whirl-Pak® Filter Bags, 6” x 9”, 24 oz.
(Stomacher® bags without mesh)
Nasco Cat. no. BO1297WA or
equivalent
Microcentrifuge tubes, PCR clean, 1.5-mL
MLS
Sterile tubes, 15- and/or 50-mL
MLS
Reagents
Buffered Peptone Water (BPW) Enrichment Broth
12
MLS
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
Chapter 2 PrepSEQ® Nucleic Acid Extraction Kit: Salmonella spp.
Pre-enrichment of primary production stage samples
2
Equipment, consumables, and reagents
Item
Source
Tetrathionate (TT) Broth
MLS
Nuclease-free Water
Cat. no. AM9938
Ethanol, 95%
MLS
Isopropanol, 100%
• Fisher Chemical Cat. no. A464-1
• Sigma-Aldrich Cat. no. 190764-1L
• Or equivalent
Pre-enrichment of primary production stage samples
Homogenization
and primary
enrichment
1. Combine sample with Tetrathionate Broth (TT Broth) at a 1:9 ratio in a filter
stomacher bag (Nasco WhirlPak Cat. no. B01488WA or equivalent); for example,
combine 25 grams of sample with 225 mL of TT Broth.
2. Homogenize the sample in a Stomacher® 400 Laboratory Blender (or equivalent)
for approximately 1 minute under normal speed.
3. Incubate the sample at 37±1°C for 16–20 hours under static conditions.
Secondary
enrichment
1. Dilute the enriched sample with Buffered Peptone Water (BPW) at a 1:9 ratio; for
example, 1 mL of sample with 9 mL of BPW in an appropriate container.
2. Incubate the sample at 37±1°C for 5±1 hour under static conditions.
3. Briefly mix the sample to ensure bacteria are in solution; for example, vortex for
about 5 seconds.
4. Proceed to sample preparation.
Note: It is recommended that BPW samples be retained for confirmation of
presumptive positives. Please store samples cold at 5±3°C.
Automated sample preparation using PrepSEQ® Nucleic Acid
Extraction Kit
IMPORTANT! Use proper aseptic technique while handling samples to avoid crosscontamination.
Prepare PrepSEQ®
kit materials
1. Set the block heater temperature to 37°C.
2. Binding Solution (Isopropanol): Add 35 mL of 100% isopropanol to the empty
Binding Solution bottle. Label the bottle to indicate that isopropanol has been
added.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
13
2
Chapter 2 PrepSEQ® Nucleic Acid Extraction Kit: Salmonella spp.
Automated sample preparation using PrepSEQ® Nucleic Acid Extraction Kit
3. Wash Buffer: Add 74 mL of 95% ethanol to a Wash Buffer Concentrate bottle, mix
well, then label the bottle to indicate that ethanol has been added.
4. Magnetic Particles: Incubate the Magnetic Particles tube at 37±1°C for
8±2 minutes, then vortex for approximately 10 seconds; keep at room temperature
(23±5°C) until ready for use. If after the initial incubation, the white precipitate is
not completely dissolved, then longer incubation and higher temperatures (up to
50°C) can be applied towards the goal of dissolving the precipitate.
IMPORTANT! White precipitate occasionally forms in the Magnetic Particles tube.
Extraction experiments show that formation of precipitate does not affect
performance as long as the precipitate is dissolved prior to use and the Magnetic
Particles resuspended. Before using, always incubate the Magnetic Particles tube
at 37±1°C for 10 minutes, then vortex to completely resuspend. If after 10 minutes
the white precipitate is not completely dissolved, then longer incubation times
and higher temperatures (≤50°C) followed by vortexing are recommended to
ensure complete resuspension of particles.
Note: The Magnetic Particles are the limiting reagent in the PrepSEQ® Nucleic
Acid Extraction Kit. Make sure that there are sufficient Magnetic Particles for the
number of samples to be processed; 25 µL of magnetic beads per sample are
required.
5. Binding Premix Solution:
a. Premix 450 µL of PK Buffer with 325 µL of Binding Buffer and 25 µL of
Magnetic Particles per sample in an appropriate container.
Note: When preparing the Binding Premix Solution for multiple samples,
multiply the volumes by the number of samples (including controls) plus an
additional 10% for sampling variation.
Note: Use the Binding Premix Solution within 1 hour of preparation. Store at
room temperature.
b. Mix well by vortexing for approx. 5 seconds until resuspension is complete.
c. Add 800 µL of Binding Premix Solution to each well of the Lysis (sample)
Plate.
d. Load the plate into the instrument when instructed by the MagMAX™
Express-96 magnetic particle processor.
Prepare the
MagMAX™
Express-96
processing plates
1. Prepare the processing plates as described in the table below. Add reagents to the
plates as described under “Action”.
Plate
Tip Plate
14
MagMAX™
Express-96 plate
type
Standard
Action
Place a 96-well Deep Well Tip Comb
into a MagMAX Express-96 standard
plate.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
Chapter 2 PrepSEQ® Nucleic Acid Extraction Kit: Salmonella spp.
Automated sample preparation using PrepSEQ® Nucleic Acid Extraction Kit
Plate
Elution Plate
MagMAX™
Express-96 plate
type
Standard
2
Action
Add 120 µL Elution Buffer per well.
Note: To include the Elution Buffer
Control, add 120 µL of Elution Buffer to
an extra empty well in the Elution Plate.
Run samples on
the MagMAX™
Express-96
instrument
Wash Plate I
Deep well
(Cat. no. 4388476)
Add 300 µL Wash Buffer.
Wash Plate II
Deep well
Add 300 µL Wash Buffer.
Lysis (sample) Plate
Deep well
Add 100 µL pre-enriched BPW liquid
sample (from step 4) from the filtered
side of the enriched sample culture
bag(s) to the Lysis (sample) Plate
containing 800 µL of Binding Premix.
1. Select the 4428176DWPrepSEQFA program on the MagMAX™ Express-96
Magnetic Particle Processor. Press Start.
2. Load the plates into the MagMAX™ Express-96 instrument according to the
readout. Verify each plate orientation {A1 to A1}.
Plate
Action
Tip Plate
Load the Tip Plate, and press Start.
Elution Plate
Load the prepared Elution Plate, and press Start.
Wash Plate II
Load the prepared Wash Plate II, and press Start.
Wash Plate I
Load the prepared Wash Plate I, and press Start.
Lysis (sample) Plate
Load the Lysis (sample) Plate, and press Start.
Note: There is no further manual interaction step with the automated run until
the run has completed the purification process.
3. When the sample preparation run is complete, the message “Enjoy your DNA” is
displayed on the screen. Remove the Elution plate.
Note: The Elution Plate contains the purified DNA.
Proceed with the MicroSEQ® Salmonella spp. real-time PCR run (as
directed) or seal the Elution Plate with clear adhesive film and store at either 5±3°C
(overnight) or below –18°C (long-term).
STOPPING POINT
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
15
2
Chapter 2 PrepSEQ® Nucleic Acid Extraction Kit: Salmonella spp.
Troubleshooting
Troubleshooting
Observation
Inhibition of PCR is
indicated by nondetection of IPC reaction.
Possible cause
Magnetic Particles were in the
elution plate.
Recommended action
Avoid disturbing the Magnetic Particles during transfer
of eluted DNA to the lyophilized assay.
Optional:
Spin the plate at 4000 × g for about 30 seconds to pellet
the Magnetic Particles to the bottom of the plate.
or
Place the elution plate on the 96-well magnetic ring
stand (Cat. no. AM10050) during transfer of sample to
the lyophilized assay.
Elution plate contains incompletely
removed particulate residue from
food sample.
Avoid residue during transfer of eluted DNA to
lyophilized assay.
Optional:
Spin the plate at 4000 × g for about 30 seconds to pellet
the food residue to the bottom of the plate.
16
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
3
PrepSEQ® Rapid Spin Sample
Preparation Kit – Extra Clean with
Proteinase K: Salmonella spp.
Overview
The PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean with Proteinase K
provides a simple way to prepare DNA from primary production stage environmental
samples for 100 reactions. The PrepSEQ® Kit is validated for use with primary
production stage environmental samples. The kit procedure involves:
• Salmonella spp. enrichment; primary enrichment in Tetrathionate Broth (TT Broth)
followed by dilution and secondary enrichment in Buffered Peptone Water (BPW)
• Sample preparation using spin columns
The recommended starting samples are supported:
• Boot socks and sponges: 100 to 150 mL of TT Broth
• Swabs: 10 mL of TT Broth
• Other environmental samples (such as feces): TT Broth at a 1:10 ratio; for example,
25 g of sample with 225 ml of TT Broth
Note: For sample preparation from enriched primary production samples,
Applied Biosystems recommends a 750-µL sample volume (loaded onto the spin
column).
Kit contents
The PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean with Proteinase K
(Cat. no. 4426715) contains reagents for 100 sample preparations.
Item
Quantity or volume
Storage
Spin columns
100
Microcentrifuge tubes, 1.5 mL
2 x 100
Lysis Buffer, 1 bottle
5 mL
5±3°C
Proteinase K (20 mg/mL), 1 tube
1.25 mL
Below –18°C
Room temperature (23±3°C)
Note: Parts may ship separately depending on the configuration ordered and storage
conditions.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
17
3
Chapter 3 PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean with Proteinase K: Salmonella spp.
Materials not included
Materials not included
The following table includes materials and equipment for using (but not included in)
the PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean with Proteinase K.
Unless otherwise indicated, all materials are available from Life Technologies. MLS:
Major laboratory supplier.
Equipment, consumables, and reagents
Source
Item
Equipment
Block heater, 97°C
MLS
Block heater, 56°C
MLS
Rack for 1.5-mL tubes
MLS
Benchtop microcentrifuge
Eppendorf 5415 D or equivalent
Homogenizer
Blender)
(Stomacher®
400 Laboratory
Seward Cat. no. 0400/001/AJ or
equivalent
MLS
Pipettors:
• Positive-displacement
• Air-displacement
Vortexer
MLS
Consumables
Disposable gloves
MLS
Aerosol-resistant pipette tips
MLS
Whirl-Pak®
Filter Bags, 10” x 15”, 92oz.
(Stomacher® bags with mesh)
Nasco Cat. no. BO1488WA or
equivalent
Whirl-Pak® Filter Bags, 6” x 9”, 24 oz., 250/pkg
(Stomacher® bags with mesh)
Nasco Cat. no. BO1348WA or
equivalent
Whirl-Pak® Bags, 6” x 9”, 24 oz.
(Stomacher® bags without mesh)
Nasco Cat. no. BO1297WA or
equivalent
Reagents
18
Buffered Peptone Water (BPW)
MLS
Tetrathionate (TT) Broth
MLS
Nuclease-free Water
Cat. no. AM9938
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
Chapter 3 PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean with Proteinase K: Salmonella spp.
Pre-enrichment of primary production stage samples
3
Pre-enrichment of primary production stage samples
Before you begin
Preparation of sample preparation reagents
• Set one block heater temperature to 97°C and set a second block heater
temperature to 56°C.
• Label 1.5-mL microcentrifuge tubes.
• For processing multiple samples we recommend preparing a Proteinase K-Lysis
Buffer mix: premix 5 µL of Proteinase K (20 mg/mL) with 50 µL of Lysis Buffer for
each sample (use a clean appropriately-sized container for mixing). Multiply
volumes by the number of samples plus 10% for overage. Mix well to disperse
Proteinase K in Lysis Buffer. Use immediately, or store on ice until ready to use.
Homogenization
and primary
enrichment
1. Combine sample with Tetrathionate Broth (TT Broth) at a 1:9 ratio in a filter
stomacher bag (Nasco WhirlPak Cat. no. B01488WA or equivalent); for example,
combine 25 grams of sample with 225 mL of TT Broth.
2. Homogenize the sample in a Stomacher® 400 Laboratory Blender (or equivalent)
for approximately 1 minute under normal speed.
3. Incubate the sample at 37±1°C for 16–20 hours under static conditions.
Secondary
enrichment
1. Dilute the enriched sample with Buffered Peptone Water (BPW) at a 1:9 ratio; for
example, 1 mL of sample with 9 mL of BPW in an appropriate container.
2. Incubate the sample at 37±1°C for 5±1 hour under static conditions.
3. Briefly mix the sample to ensure bacteria are in solution; for example, vortex for
about 5 seconds.
4. Proceed to sample preparation.
Note: It is recommended that BPW samples be retained for confirmation of
presumptive positives.
Sample preparation using PrepSEQ Rapid Spin Sample Preparation
Kit – Extra Clean with Proteinase K
IMPORTANT! Use proper aseptic technique while handling samples to avoid crosscontamination.
1. Insert a spin column into a labeled tube.
2. Load 750 µL of your enriched sample onto the spin column and cap the column.
3. Load the tube with its spin column into the microcentrifuge. Place the tube cap
hinge toward the inside of the rotor (see figure below, at right). Otherwise the cap
hinge interferes with installation of the rotor lid.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
19
3
Chapter 3 PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean with Proteinase K: Salmonella spp.
Sample preparation using PrepSEQ Rapid Spin Sample Preparation Kit – Extra Clean with Proteinase K
Incorrect position of the tube cap
Correct position of the tube cap
4. Microcentrifuge the tube for 3 minutes at 12,000–16,000 × g.
5. Remove the tube from the microcentrifuge and discard the used spin column.
6. Aspirate, then discard the supernatant.
IMPORTANT! Remove supernatant as completely as possible, including any
excess liquid on the sides of the tube. Remove droplets by circling the inside of the
tube with the pipettor and pushing into supernatant for removal by aspiration.
7. Add 55 µL of Proteinase K-Lysis Buffer mix (50 µL of Lysis Buffer + 5 µL of
Proteinase K) per sample to the pellet.
Note: See “Before you begin” on page 19 for instructions on how to prepare
Proteinase K-Lysis Buffer mix when processing multiple samples.
IMPORTANT! Store Proteinase K-Lysis Buffer mix on ice until ready to use.
8. Resuspend by pipetting up and down, or vortex until the pellet is well dispersed
in the Lysis Buffer mix.
9. Transfer the Proteinase K-Lysis Buffer mixture containing the resuspended
bacterial pellet into a clean 1.5-mL tube. The pellet must be well dispersed in the
Lysis Buffer prior to transfer.
IMPORTANT! The Proteinase K-Lysis Buffer mix needs to be transferred to a clean
tube. During the transfer there will be residual fat on the sides of the original
tube. Avoid contact with the fat and transfer only the Proteinase K-Lysis Buffer
mix containing the resuspended pellet into a clean tube.
10. Cap the tube, then incubate at 56±1°C for about 30 minutes to activate the
Proteinase K.
11. Incubate at 97±2°C for about 10 minutes.
12. Allow the sample to cool for about 2 minutes at room temperature.
13. Microcentrifuge the tube for 1 minute at 12,000–16,000 × g to bring down
condensation following the 97±2°C heating step.
20
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
Chapter 3 PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean with Proteinase K: Salmonella spp.
Sample preparation using PrepSEQ Rapid Spin Sample Preparation Kit – Extra Clean with Proteinase K
3
14. Add 250 µL of Nuclease-free Water. Mix well.
IMPORTANT! Use Nuclease-free Water (Cat. no. AM9938). Autoclaved water
should not be considered PCR-clean.
15. Microcentrifuge the tube for 90±30 seconds at 12,000–16,000 × g to bring down any
particulate material derived from the spin column, which can interfere with
amplification. The microbial DNA is in the aqueous phase.
Note: Samples can be stored at 5±3°C overnight, or below –18°C for long term
storage. Prior to running PCR, stored samples should be centrifuged for about
1 minute at maximum speed in order to pellet the column particulate material.
16. Proceed with PCR, or store the tube below –18°C.
IMPORTANT! Use 30 µL of supernatant for PCR in the lyophilized assay. For
detailed instructions, refer to the MicroSEQ® Salmonella spp. Detection Kit
Protocol (see “MicroSEQ® Salmonella spp. Detection Kit” on page 23).
IMPORTANT! If PCR inhibition occurs, as indicated by no amplification for either
target or IPC, then dilute sample with water. It is recommended to add 5 µL of
sample and 25 µL of water to the lyophilized assay to overcome inhibition. See
“Troubleshooting” on page 22 for further discussion.
For samples with high fat content, a top
layer containing debris can form over the
DNA sample. Avoid the top layer and
bottom pellet by collecting the sample for
PCR from the clear center section.
Avoid top layer containing sample debris.
Collect sample from center.
Particulate material from column.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
21
3
Chapter 3 PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean with Proteinase K: Salmonella spp.
Troubleshooting
Troubleshooting
Observation
Inhibition of PCR,
indicated by nondetection of IPC
reaction
The bacterial pellet
separates from the
tube, making the
pellet difficult to avoid
during aspiration.
22
Possible cause
Recommended action
Removal of the supernatant
before adding Lysis Buffer
was not sufficient.
Dilute the sample 1:5 or 1:10 with Nuclease-free Water to dilute
PCR inhibitors. If PCR remains inhibited, repeat the sample
preparation.
Filtrate from the spin column
is in the sample. The filtrate
containing particulate
material can inhibit PCR.
Centrifuge the sample to separate the filter particulates before
transferring sample to the PCR reaction.
Matrix associated with PCR
inhibitory components.
Pre-wash the bacterial pellet before loading the Rapid Spin
column:
Because feces are
heterogeneous and variable
between sources there is a
high probability of
complications. This step
offers a solution to help
remove PCR inhibitory
components.
1 – Transfer 750 µL of sample to a clean microcentrifuge tube.
The sample was left
unattended before aspirating
off the supernatant, causing
dissipation of the bacterial
pellet.
Re-centrifuge and remove the supernatant immediately
following centrifugation.
The size of the bacterial
pellet is very small and
difficult to see.
Remove the supernatant carefully, leaving behind up to 50 µL of
supernatant, to avoid aspiration of pellet.
2 – Centrifuge at 12,000–16,000 × g for 3 min.
3 – Discard supernatant.
4 – Resuspend pellet in 650 µL of sterile distilled water.
5 – Load the resuspended sample onto the Rapid Spin column.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
MicroSEQ® Salmonella spp.
Detection Kit
4
Overview
The MicroSEQ® Salmonella spp. real-time PCR Detection Kit has been validated for use
with primary production samples.
Kit contents
The MicroSEQ®Salmonella spp. Detection Kit (Cat. no. 4403930) contains reagents for
96 reactions. Kit components and their storage conditions are shown in the table
below.
Description
Cap color
Storage
Salmonella spp. Assay Beads, 96 tubes in 8-tube strips
(96 reactions/kit), twelve 8-tube strips
Green (rack)
MicroAmp® Optical 8-Cap Strips, twelve 8-tube strips
N/A
5±3°C;
protect from
light and
moisture‡
Pathogen Detection Negative Control, 1 tube, 1.5-mL
Red
5±3°C
Component
MicroSEQ® Salmonella spp.
Detection Kit
Pathogen Detection Negative
Control
‡ Excessive exposure to light may affect the fluorescent probes. Seal the pouch tightly each time you remove 8-tube strips from the pouch to
protect from moisture.
Note: Parts may ship separately depending on configuration and storage conditions.
Materials not included
The following table includes materials and equipment for using (but not included in)
the MicroSEQ®Salmonella spp. Detection Kit. Unless otherwise indicated, all materials
are available from Life Technologies. MLS: Major laboratory supplier.
Instruments, equipment, consumables, and reagents‡
Item
Source
Instruments
Applied
System
Biosystems®
7500 Fast Real-Time PCR
StepOne™ Real-Time PCR System
Contact your local Life Technologies
representative.
StepOnePlus™ Real-Time PCR System
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
23
4
Chapter 4 MicroSEQ® Salmonella spp. Detection Kit
Prepare for PCR
Instruments, equipment, consumables, and reagents‡ (continued)
Item
Source
Equipment
Benchtop microcentrifuge
Eppendorf 5415D or equivalent
Block heater
MLS
Ice bucket
MLS
Plate centrifuge
Eppendorf 5804 or equivalent
Vortexer
MLS
MicroAmp®
7500 Fast Precision Plate Holder for
Tube Strips (for use with 7500 Fast Real-Time PCR
System§)
Cat. no. 4403809
MicroAmp® Fast 48-Well Tray (for use
with StepOne™ Real-Time PCR System)
Cat. no. 4375282
MicroAmp® 96-Well Tray for Veriflex™ Blocks (for
use with StepOnePlus™ Real-Time PCR System)
Cat. no. 4379983
MicroAmp® 96-Well Base
Cat. no. N8010531
Pipettors:
MLS
• Positive-displacement
• Air-displacement
• Multichannel
Consumables
Aerosol-resistant pipette tips
MLS
Disposable gloves
MLS
MicroAmp® Multi-removal Tool
Cat. no. 4313950
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
‡ The materials listed here have been validated for use with this kit. Results may vary if substituted products
from other vendors are used instead.
§ included in the starter kit
Prepare for PCR
To prepare for PCR, you must create a run file document and prepare the assay beads
and samples. The exact procedure depends on whether or not you use RapidFinder™
Express Software. Life Technologies recommends using the 7500 Fast Real-Time PCR
System with the RapidFinder™ Express Software; see “StepOne™ and StepOnePlus™
Systems” on page 33 for information on running reactions using other
24
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
Chapter 4 MicroSEQ® Salmonella spp. Detection Kit
Prepare for PCR
4
instrumentation. This protocol describes the procedure that does not use
RapidFinder™ Express Software. We recommend using RapidFinder Express Software
for online step-by-step instructions to set up the assays followed by automated data
analysis.
IMPORTANT! If you use RapidFinder™ Express Software, refer to the “Create or Edit
Run File” and “Pipette Samples” instructions in the RapidFinder™ Express Software
Online Help (Pub. no. 4401842).
Prepare PCR
Create a run file document
1. In the Sequence Detection System Software, create a run file document: In the
Assay drop-down list, select Absolute Quantification (Standard Curve). For
information on creating a run file document, refer to the documentation that is
provided with your instrument.
2. With the Quencher Dye set to (none) or (Non Fluorescent), create or select FAM™
and VIC® dye detectors.
3. Associate both FAM™ and VIC® detectors with each reaction.
Note: The FAM dye is used to detect the target; the VIC dye is used to detect the
internal positive control (IPC).
4. Set thermal-cycling conditions as indicated in the table below. For more details,
refer to the 7300/7500/7500 Fast Real-Time PCR System Absolute Quantitation Using
Standard Curve Getting Started Guide (Pub. no. 4347825) or the StepOne™ and
StepOnePlus™ Real-Time PCR Systems Presence/Absence Experiments Getting Started
Guide (Pub. no. 4376787).
For 7500 Fast instruments
Step
Enzyme activation
PCR
HOLD
Cycle (40 cycles)
Denature
Anneal/extend
Temp.
95 °C
95 °C
60 °C
Time
2 min
3 sec
30 sec
For StepOne™ and StepOnePlus™ instruments
(not included in AOAC or AFNOR validation studies)
Step
Enzyme activation
PCR
HOLD
Cycle (40 cycles)
Denature
Anneal/extend
Temp.
95 °C
95 °C
60 °C
Time
2 min
1 sec
20 sec
5. Set the Sample Volume to 30 µL.
6. Select the appropriate run mode for your system according to the following table:
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
25
4
Chapter 4 MicroSEQ® Salmonella spp. Detection Kit
Prepare for PCR
System
Run mode
7500 Fast‡
Fast 7500
StepOne™
Fast
StepOnePlus™
Fast
‡ Figure 1 shows an example of the Instrument tab of the 7500 Fast System SDS Software.
Figure 1 Instrument tab for 7500 Fast System SDS Software with run mode set to Fast 7500.
Prepare the assay beads
1. Open the storage pouch containing the assay beads (cut at the notch in the upper
corner of the storage pouch above the zip-lock strip).
IMPORTANT! Do not remove the desiccant from the storage pouch.
2. Remove the appropriate number of individual tubes or 8-tube strips (one tube for
each reaction that you plan to run).
3. Place the tubes on ice or in a cold block.
26
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
Chapter 4 MicroSEQ® Salmonella spp. Detection Kit
Prepare for PCR
4
4. Seal the storage pouch using the zip-lock strip, then store at 5±3°C.
Prepare samples and controls
1. Place samples and controls on ice or in a cold block. Thaw completely.
IMPORTANT! Reagents must be thawed on ice or in a cold block.
2. Remove condensation after thawing samples and prior to opening to avoid cross
contamination.
a. For PrepSEQ® Nucleic Acid Extraction Kit samples, centrifuge the plate at
1000–2000 × g for 90±30 seconds.
b. For PrepSEQ® Rapid Spin Sample Preparation Kit samples, microcentrifuge
the tubes at 12,000–16,000 × g for 90±30 seconds to bring down any
particulate material derived from the spin column, which can interfere with
amplification. The microbial DNA is in the aqueous phase.
c. For Pathogen Detection Negative Control and positive control(s) in
microcentrifuge tubes, vortex and then spin down the tubes.
Note: Unknown samples and positive control samples are provided by the
investigator. The kit includes a negative control (Pathogen Detection Negative
Control).
3. Add 30 µL of sample prepared above to each assay bead. Dispense all unknown
samples first, followed by negative control(s), and then positive control(s). Beads
dissolve in 1 to 5 seconds. Mix by gently aspirating and dispensing a few times.
(Alternatively, vortex the assay tubes after they are capped in the final step.)
IMPORTANT! Use a new pipette tip for each sample. Resuspend by gently
pipetting up and down with the pipette tip at the bottom of the tube to minimize
aerosol formation and cross-contamination or, if a vortexer is available, vortex the
capped tubes until the pellet is dissolved.
Note: Add 30 µL of the PrepSEQ® Rapid Spin Kit sample, or 30 µL of the
PrepSEQ® Nucleic Acid Extraction Kit sample, to each assay bead. The PrepSEQ®
Rapid Spin Kit provides 300 µL of sample, and the PrepSEQ® Nucleic Acid
Extraction Kit provides ~100 µL of sample. Use 30 µL of the Pathogen Detection
Negative Control per negative control reaction. If you use other sample
preparation methods, and less than 30 µL of sample is available, adjust the
sample volume with water to 30 µL for each reaction type.
IMPORTANT! Cap the tubes, sealing each tube with the transparent optical strip
caps provided in the kit. Do not use colored caps or tubes for kit reactions.
Colored caps or tubes may affect dye-signal readings during real-time PCR.
Note: The assay beads contain all the components necessary for each reaction.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
27
4
Chapter 4 MicroSEQ® Salmonella spp. Detection Kit
Run the reactions
Prepare tubes for
the 7500 Fast,
StepOne™, and
StepOnePlus™
Systems
The 8-tube strips (containing assay beads) provided are compatible with the 7500 Fast,
the StepOne™, and StepOnePlus™ Systems. For information on using the StepOne™
and StepOnePlus™ Systems, see Appendix A on page 33.
1. For 8-tube strips with seven or fewer reactions, add additional empty tubes as
needed so that each strip contains a full set of 8 tubes.
Note: The 8-tube strips evenly distribute the clamping load that is applied to the
sample tube strips during processing and will therefore minimize the risk of
collapsing any tubes. Add empty tubes as needed to make a full strip of 8 tubes in
a column (see figure on next page).
2. Cap the tubes, sealing each tube with the transparent optical strip caps provided
in the kit. Cap the tubes with the strip caps using the MicroAmp® 96-Well Base
(Cat. no. N8010531) and the MicroAmp® Cap Installing Tool (Cat. no. 4330015) to
avoid collapsing, bending, or misaligning the tubes. Confirm that the strips are
straight and that each tube is in line with the adjacent tube.
3. Make sure reactions are thoroughly mixed.
IMPORTANT! Mixing can be accomplished by gentle pipetting up and down.
Keep the pipette tip at the bottom of the tube to minimize aerosol formation and
cross-contamination, or, if available, use a vortexer to mix the assay tube
components.
4. Make sure reagents are at the bottom of the tubes. If available, spin down the tube
contents at 2000 × g for about 20 seconds using a centrifuge with a plate adapter.
Run the reactions
Running tubes or a plate involves using an Applied Biosystems® Sequence Detection
System (SDS) or Real-Time PCR System for sample analysis. The exact procedure
depends on whether or not you use RapidFinder™ Express Software. This protocol
describes the procedure that does not use RapidFinder Express Software. We
recommend using RapidFinder™ Express Software for online step-by-step instructions
to set up the assays followed by automated data analysis.
IMPORTANT! If you use RapidFinder™ Express Software, refer to the “Start Instrument
Run” instructions in the RapidFinder™ Express Software Online Help (Pub. no. 4401842).
Before you begin
Ensure that your instrument is properly installed and calibrated. For calibration
information, see the documentation that is provided with your instrument.
Run the 8-tube
reactions on the
7500 Fast System
(only) without
RapidFinder™
Express Software
For 8-tube strips on the 7500 Fast System:
28
Use of the 7500 Fast Precision Plate Holder for MicroAmp® Tube Strips
(Cat. no. 4403809) is recommended.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
Chapter 4 MicroSEQ® Salmonella spp. Detection Kit
Run the reactions
4
When you use the MicroAmp 8-tube strips, if column 1 (left most) and column 12
(right most) are not used, insert two fully capped, empty, 8-tube strips into these
columns.
Note: The empty capped 8-tube strips evenly distribute the clamping load applied to
the sample tube strips during processing, thereby minimizing the risk of collapsing
any tubes.
1. Carefully insert two or more 8-tube strips containing samples, starting from the
center of the plate holder and moving out. This layout minimizes bending or
misaligning the tube strips.
Note: A minimum of two and a maximum of twelve 8-tube strips can be run at
one time. If your samples are in an odd number of strip tubes, you will need to
include an empty capped 8-tube strip to balance the load.
IMPORTANT! Always use a total of 8 tubes per column. You may need to add new,
empty tubes to a column.
2. Open the run file document that corresponds to the reaction plate that you
created in “Create a run file document” on page 25.
3. Start the run.
IMPORTANT! To avoid false positives due to amplified material in your work area,
do not open tubes after amplification.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
29
4
Chapter 4 MicroSEQ® Salmonella spp. Detection Kit
View results
View results
Overview
How you view results depends on the instrument you use. Refer to the appropriate
user guide of your real-time PCR instrument or the RapidFinder™ Express Software
Online Help for instructions on how to analyze data and view your results.
IMPORTANT! If you use RapidFinder™ Express Software, refer to the “Viewing
Results” instructions in the RapidFinder™ Express Software Online Help
(Pub. No. 4401842).
General process
without
RapidFinder™
Express Software
The general process for viewing results from MicroSEQ® Pathogen Detection Kits
involves:
• Viewing the amplification plots for all reactions.
• Setting the baseline and threshold values.
• Checking each sample for a FAM™ dye (target-specific) signal and a VIC® dye
(IPC) signal. The following table is a basic guide for interpreting the results:
Resources for
viewing results
FAM™ dye signal
(target)
VIC® dye signal
(IPC)
+
+, –
Positive
–
+
Negative
–
–
See “Troubleshooting” on page 32
Result
For more information about analyzing your data, refer to the:
• Appropriate instrument user guide
• RapidFinder™ Express Software Online Help (Pub. no. 4401842)
We do not recommend using the same method to screen samples and confirm the
results. When you use the MicroSEQ® Pathogen Detection System to screen samples,
culture and biochemical methods are recommended to confirm the result. Refer to the
enriched BPW sample (“Secondary enrichment” on page 13 and follow ISO 6579:2002
(E), an approved protocol for confirmation of culture testing (see “References” on
page 43).
30
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
Chapter 4 MicroSEQ® Salmonella spp. Detection Kit
View results
Confirmation of
results
4
In the context of NF Validation, all samples identified as positive by the MicroSEQ®
Salmonella spp. method must be confirmed, from the BPW enrichment broth, by
performing one of the following tests:
• According to classical tests described in methods standardized by CEN ISO or
NF U47-100. The confirmation step must start from the BPW enrichment broth.
• Perform a second enrichment step in Rappaport-Vassiliadis Soya Peptone (RVS)
broth (0.1 mL BPW in 10 mL of RVS broth): incubate at 41.5±1°C for 24±3 h and
streak onto Xylose Lysine Deoxycholate (XLD) agar or another selective agar
plate. Then either perform a Latex test (Oxoid™ FT0203A) on the observed
characteristic colonies, or perform a biochemical gallery on purified colonies
characteristic of Salmonella.
• Any other method certified by “NF Validation” that is based on a different
principle than the MicroSEQ® Salmonella spp. method. It is necessary that the
complete protocol of the second validated method be performed entirely, which
means that all steps that precede the confirmation step must be common to both
methods.
In the event of discordant results (positive with the alternative method, not confirmed
by one of the means described above), the laboratory must follow the necessary steps
to guarantee the validity of the obtained result.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
31
4
Chapter 4 MicroSEQ® Salmonella spp. Detection Kit
Troubleshooting
Troubleshooting
Observation
No signal (IPC or target-specific) is
detected in unknown wells.
Possible cause
Inhibition of PCR occurred.
Action
Dilute the sample 1:5 or 1:10 with
Nuclease-free Water to dilute PCR
inhibitors and repeat the assay. If
PCR remains inhibited, repeat the
sample preparation.
For more information, refer to
“Troubleshooting” section of the
PrepSEQ® Nucleic Acid Extraction
Kit Protocol on page 16, PrepSEQ®
Rapid Spin Sample Preparation Kit
Protocol on page 22.
No target-specific signal is
detected in positive control wells.
A pipetting error occurred (no positive
control was added).
Repeat the assay. Make sure to
pipette positive control into all
positive-control wells.
No IPC is detected, but targetspecific signal is detected.
A high copy number of target DNA exists in
samples, resulting in preferential
amplification of the target-specific DNA.
No action is required.
Target-specific signal is detected in
negative-control wells
Carryover contamination occurred.
Repeat the assay using fresh
aliquots of all reagents and clean
pipetting equipment.
If the negative control continues to
show contamination, repeat the
assay using a new kit.
If the negative control continues to
show contamination, contact
Applied Biosystems Technical
Support.
In negative-control wells, a targetspecific signal is detected, but no
IPC signal is detected.
Carryover contamination and one of the
following occurred:
• A high copy number of target DNA exists
in samples, resulting in preferential
amplification of the target-specific DNA
• A problem occurred with IPC
amplification
No IPC signal is detected, but
target-specific signal is detected in
positive-control wells.
32
A high copy number of target DNA exists in
samples, resulting in preferential
amplification of the target-specific DNA.
Examine unknowns to determine if
an IPC signal is present. If an IPC
signal is present in unknown wells,
IPC amplification is not a problem.
Repeat the assay using fresh
aliquots of all reagents and clean
pipetting equipment.
No action is required.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
A
StepOne™ and StepOnePlus™
Systems
Run the 8-tube reactions on the StepOne™ System
1. Place the MicroAmp® Fast 48-Well Tray (Cat. no. 4375282) on the sample block of
the StepOne™ System.
2. Load the 8-tube strips horizontally. For example, in Row C, load an 8-tube strip
across columns 1 through 8. A minimum of one 8-tube strip is recommended. It is
not necessary to balance the tube strips on the tray.
3. Open the run file document that corresponds to the reaction plate that you
created in “Create a run file document” on page 25.
4. Start the run.
IMPORTANT! To avoid false positives due to amplified material in your work area,
do not open tubes after amplification.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
33
A
Appendix A StepOne™ and StepOnePlus™ Systems
Run the 8-tube reactions on the StepOnePlus™ System
Run the 8-tube reactions on the StepOnePlus™ System
1. Place the MicroAmp® 96-Well Tray for Veriflex™ Blocks (Cat. no. 4379983) on the
sample block of the StepOnePlus System.
2. Load the 8-tube strips vertically. For example, load the 8-tube strips in columns 1
and 2 across rows A through H in both columns. The minimum recommended
load is two 8-tube strips (16 tubes), which you should place in adjacent columns,
for example in columns 1 and 2. It is not necessary to balance the tube strips on
the tray.
3. Open the run file document that corresponds to the reaction plate that you
created in “Create a run file document” on page 25.
4. Start the run.
IMPORTANT! To avoid false positives due to amplified material in your work area,
do not open tubes after amplification.
34
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
B
Good PCR practices
Prevent contamination and nonspecific amplification
Overview
PCR assays require special laboratory practices to avoid false positive amplifications.
The high throughput and repetition of these assays can lead to amplification of a single
DNA molecule.
PCR good
laboratory
practices
When preparing samples for PCR/RT-PCR amplification:
• Wear clean gloves and a clean lab coat (not previously worn while handling
amplified products or used during sample preparation).
• Change gloves whenever you suspect that they are contaminated.
• Maintain separate work areas, dedicated equipment, and supplies for:
– Sample preparation and setup.
– Amplification and analysis of products.
• Do not bring amplified products into the reaction setup area.
• Open and close all sample tubes carefully. Avoid splashing or spraying 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).
IMPORTANT! To avoid false positives due to cross-contamination:
· Prepare and close all negative-control and unknown sample tubes before pipetting
the positive control.
· Do not open tubes after amplification.
· Use different sets of pipettors when pipetting negative-control, unknown, and
positive-control samples.
Plate layout
suggestions
• Separate different targets by a row if enough space is available.
• Put at least one well between unknown samples and controls if possible.
• Separate negative and positive controls by one well if possible.
• Place replicates of one sample for the same target next to each other.
• Start with the unknown samples and put controls at the end of the row or column.
• Put positive controls in one of the outer rows or columns if possible.
• Consider that caps come in strips of 8 or 12.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
35
B
36
Appendix B Good PCR practices
Prevent contamination and nonspecific amplification
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
C
Safety
WARNING! GENERAL SAFETY. Using this product in a manner not specified in the
user documentation may result in personal injury or damage to the instrument or
device. Ensure that anyone using this product has received instructions in general
safety practices for laboratories and the safety information provided in this document.
· Before using an instrument or device, read and understand the safety
information provided in the user documentation provided by the
manufacturer of the instrument or device.
· Before handling chemicals, read and understand all applicable Safety Data
Sheets (SDSs) and use appropriate personal protective equipment (gloves,
gowns, eye protection, etc). To obtain SDSs, see the “Documentation and
support” section in this document.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
37
C
Appendix C Safety
Chemical safety
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure
laboratory personnel read and practice the general safety guidelines for chemical
usage, storage, and waste provided below, and consult the relevant Safety Data Sheet
(SDS) for specific precautions and instructions:
· Read and understand the SDSs provided by the chemical manufacturer
before you store, handle, or work with any chemicals or hazardous
materials. To obtain SDSs, see the “Documentation and support” section in
this document.
· Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective 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, follow
the 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 spills
or leaks from the primary container. Both containers must be compatible
with the waste material and meet federal, state, and local requirements for
container 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.
38
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
Appendix C Safety
Biological hazard 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 to transmit
infectious diseases. All work should be conducted in properly equipped facilities using
the appropriate safety equipment (for example, physical containment devices). Safety
equipment also may include items for personal protection, such as gloves, coats,
gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles.
Individuals should be trained according to applicable regulatory and company/
institution requirements before working with potentially biohazardous materials.
Follow all applicable local, state/provincial, and/or national regulations. The following
references provide general guidelines when handling biological samples in laboratory
environment.
· U.S. Department of Health and Human Services, Biosafety in
Microbiological and 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
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
39
C
40
Appendix C Safety
Biological hazard safety
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
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, contact the
chemical manufacturer.
Obtaining Certificates of Analysis
The Certificate of Analysis provides detailed quality control and product qualification
information for each product. Certificates of Analysis are available on our website. Go
to www.lifetechnologies.com/support and search for the Certificate of Analysis by
product 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 or www.lifetechnologies.com/foodsafety
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
• Search for user documents, SDSs, vector maps and sequences, application notes,
formulations, handbooks, certificates of analysis, citations, and other product
support 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 (Inside North America): 1-800-500-6885
Phone number (Outside North America): Go to www.lifetechnologies.com/
contactus.html and select the appropriate country from the drop-down menu.
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
41
Documentation and support
Limited product warranty
Limited product warranty
Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth
in the Life Technologies' General Terms and Conditions of Sale found on Life
Technologies’ website at www.lifetechnologies.com/termsandconditions. If you have
any questions, please contact Life Technologies at www.lifetechnologies.com/support.
42
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
References
Association of Analytical Communities (AOAC). www.aoac.org. 2010. Accessed
November 2010.
Association Francaise pour la Normalisation (AFNOR). www.afnor-validation.com.
2010. Accessed November 2010.
ISO 6579:2002 (E). Microbiology of food and animal feeding stuffs – Horizontal
method for the detection of Salmonella spp.
ISO 6887, parts 1 through 4. Microbiology of food and animal feeding stuffs –
Preparation of test samples, initial suspension and decimal dilutions for
microbiological examination.
ISO 7218:2007. Microbiology of food and animal feeding stuffs – General requirements
and guidance for microbiological examinations.
ISO 16140:2003. Microbiology of food and animal feeding stuffs – Protocol for the
validation of alternative methods.
NF EN ISO 6579:2002/Amd 1:2007: Microbiology of food and animal feeding stuffs.
Horizontal method for the detection of Salmonella spp. Detection of Salmonella spp. in
animal faeces and in environmental samples from the primary production stage
NF U47-100: French norm on isolation and identification of Salmonella spp. in the
environment and samples from animal productions: "Recherche par l’isolement et
l’identification de tout sérovar ou de sérovar(s) spécifié(s) de salmonelles dans
l’environnement des productions animales (Detection by streaking of any Salmonella
serotypes in primary production samples)
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
43
References
44
Real-Time PCR Detection of Salmonella spp. in Primary Production Samples
For support visit www.lifetechnologies.com/support or email [email protected]
www.lifetechnologies.com
24 September 2014