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USER GUIDE
For testing of Food and Environmental samples only.
PrepSEQ® Nucleic Acid Extraction Kit:
Listeriaspp.
Preparation of MicroSEQ® PCR-ready DNA from food
and environmental samples
For use with: MagMAX™ Express-96 Deep Well Magnetic Particle Processor
Catalog Numbers 4428176, 4480466
Publication Number 4426514
Revision C
The information in this guide is subject to change without notice.
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INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT
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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.
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© 2013 Life Technologies Corporation. All rights reserved.
Contents
3
PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
About this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
■CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Kit contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Materials not included in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
■CHAPTER 2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria spp. . . . 11
Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Kit workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Sample enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Enrich of food sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Enrich of environmental sample using a swab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Enrich of environmental sample using a sponge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
DNA isolation (Proteinase K lysis protocol) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
■APPENDIX A Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . 18
Background information and product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Description of target microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Kit sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Operating conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
AOAC® Performance Tested MethodsSM Certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Confirmation of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
■APPENDIX B Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Specific chemical handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Contents
Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Obtaining Certificates of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Food safety support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
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PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Contents
6PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
About this guide
IMPORTANT! Before using the products described in this guide, read and understand
the information in the “Safety” appendix in this document.
Revision history
Revision Date Description
C November 2013 • Added certification details for AOAC® Performance Tested MethodsSM
certification.
• Updated number format (time, temperature, and centrifugation speeds) for
AOAC® certification.
• Updated to a Life Technologies template with associated updates to the
limited license information, warranty information, trademark statement, and
safety statements.
B March 2010 Baseline for revision history.
1
7
PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Product information
Ensure that your instrument is properly installed and calibrated. For calibration
information, see the documentation that is provided with your instrument.
Overview
Use the PrepSEQ® Nucleic Acid Extraction Kit to prepare food and environmental
samples to test for Listeria spp. The kit procedure involves:
• Enrichment of food and environmental samples for Listeria spp.
• Nucleic acid extraction
To extract DNA from Listeria spp. in food and environmental samples, the
Proteinase K lysis protocol that is described on page 14 is recommended.
For the isolation and purification of DNA from enriched food and environmental
samples, a 1-mL sample volume is recommended. The recommended elution volume
is 140 µL.
Refer to Appendix A, “Supplemental information” for detailed information about
AOAC® certification.
The PrepSEQ® Nucleic Acid Extraction Kit is for professional use only. Users may
include, but are not limited to, food producers, food processors, food manufacturers,
retailers, and microbiology testing laboratories.
Visit www.lifetechnologies.com/foodsafety for a list of workflows for detection of
Listeria spp.
8PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Chapter1 Product information
Kit contents
1
Kit contents
Table1 PrepSEQ® Nucleic Acid Extraction Kit
Note: Parts may ship separately depending on configuration and storage conditions.
Materials not included in the kit
The materials listed here have been validated for use with this kit. Results may vary if
substituted products from other vendors are used instead. Unless otherwise indicated,
many of the listed items are available from major laboratory suppliers (MLS).
Item Cat. no.4480466
(100 reactions)‡
‡ Cat. no.4445658 also includes the MicroSEQ® Listeria spp. Detection Kit.
Cat. no. 4428176
(300 reactions) Storage
Magnetic Particles 1 tube, 1.5 mL/tube 6 tubes, 1.5 mL/tube 5±3°C
Lysis Buffer 1 bottle, 50 mL/bottle 6 bottles, 50 mL/bottle
Room temperature
(23±5°C)
Binding Solution (Isopropanol) 1 empty bottle 3 empty bottles
Wash Buffer Concentrate 2 bottles, 26 mL/bottle 6 bottles, 26 mL/bottle
Elution Buffer 1 bottle, 25 mL 3 bottles, 25 mL
Proteinase K (PK) Buffer 1 bottle, 50 mL 3 bottles, 50 mL
Proteinase K (20 mg/mL) 1 tube, 1.25 mL 3 tubes, 1.25 mL Below –18°C
Item Source
Equipment
Benchtop microcentrifuge Eppendorf 5415 D or equivalent
MagMAX™ Express-96 Deep Well Magnetic
Particle Processor
Cat. no. 4400079
96 Well Magnetic-Ring Stand Cat. no. AM10050
Homogenizer (Stomacher® 400 Laboratory
Blender or equivalent)
Seward #0400/001/AJ or equivalent
Vortexer MLS
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PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Chapter1 Product information
Materials not included in the kit 1
Consumables
Disposable gloves MLS
Micropipette tips, aerosol-resistant MLS
Pipettors:
• Positive-displacement
• Air-displacement
• Multichannel
MLS
15-mL conical tubes MLS
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
Swab, cotton MLS
Microcentrifuge tubes, PCR clean, 1.5-mL MLS
Homogenizer bags appropriate for your sample:
With sponge, 4.5” × 9” (Whirl-Pak® Speci-Sponge
Environmental Sampling Bag, or equivalent)
Nasco # B01245WA or equivalent
With mesh, 6” × 9”, 24 oz (Whirl-Pak® Filter Bag
for Homogenizer Blender, or equivalent)
Nasco # B01348WA or equivalent
No mesh, 6” × 9”, 24 oz (Whirl-Pak® Write-on
Bag, or equivalent)
Nasco # B01196WA or equivalent
Reagents
Ethanol, 95% MLS
Isopropanol, 100% MLS
Dey Engley (D/E) Neutralizing Broth BD # 281910, or equivalent
Buffered Listeria Enrichment Broth (BLEB), 500 g VWR # EM1.09628.0500
Listeria Selective Enrichment Supplement,
16 × 1 mg/vial
VWR # EM1.11781.0001
Nuclease-free water Cat. no. AM9938
Item Source
10 PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Chapter1 Product information
Materials not included in the kit
1
2
11
PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
PrepSEQ® Nucleic Acid Extraction
Kit: Listeria spp.
Before you begin
Before starting your sample extraction:
•Binding Solution – Add 35 mL of 100% isopropanol to the 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,
mix well, then label the bottle to indicate that ethanol is added.
•Magnetic Particles – Incubate the Magnetic Particles tube at 37±1°C for about
10 minutes, then vortex until the beads are completely resuspended (at least
10 seconds), then keep at room temperature (23±5°C) until ready for use.
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. If a precipitate
forms, incubate the tube at 37±1°C for about 10 minutes. If after ten minutes the
white precipitate is not completely dissolved, then you may apply longer
incubation and higher temperatures (up to 50°C). Then vortex to completely
resuspend.
Note: The magnetic particles are the limiting reagent in the PrepSEQ® Nucleic
Acid Extraction Kit. Make sure you have enough magnetic particles for the
number of samples you will process. 30 µL of magnetic beads are required per
sample.
•Proteinase K Buffer mix – For processing multiple samples, we recommend
preparing a Proteinase K Buffer mix:
a. Premix 10 µL of Proteinase K (20 mg/mL) with 200 µL of PK Buffer for each
sample (use a clean appropriately-sized container for mixing).
b. Multiply volumes by the number of samples plus 10% for overage.
c. Mix well to disperse Proteinase K in Proteinase K Buffer. Use immediately or
store on ice until ready to use.
Kit workflow
Below is a sample preparation workflow based on the Proteinase K lysis protocol;
detailed procedures start on page 13.
12 PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Chapter2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria spp.
Kit workflow
2
Step 1: Transfer 1 mL to a 1.5-mL tube.
Proteinase K lysis protocol
Step 2: Centrifuge the tube at 12,000–16,000 × g for about 3 minutes.
Step 5: Transfer the sample to a MagMAX™ Express-96 Deep Well Plate (lysis plate).
Step 7: Select 44000799DWPrepSEQGP from the MagMAX™ Express-96 magnetic particle processor.
Step 8a–e: Load plates into the MagMAX™ Express-96 magnetic particle processor, then press Start.
Step 10: After 18 min, dispense the Binding Mix:
a. Vortex Magnetic Particles for at least 10 sec.
b. Add 30 µL of Magnetic Particles to each well of the lysis plate.
c. Add 300 µL of Binding Solution to each well of the lysis plate.
d. Load the plate into the instrument, then press Start.
Step 3: Remove and discard the supernatant as quickly as possible.
Step 9: After 20 min, dispense the Lysis Buffer:
a. Add 300 µL of Lysis Buffer to each well of the lysis plate.
b. Load the plate into the instrument, then press Start.
Step 4: Add 210 µL of Proteinase K Buffer mix (10 µL PK + 200 µL PK Buffer). Mix well to resuspend the pellet.
Step 11: After 30 min, sample preparation is done. The message “Enjoy your DNA” is displayed
on the screen. Remove the elution plate. Proceed with PCR, or seal the plate and store below –18°C.
Step 6a–b: Prepare the elution and wash plates (MagMAX™ Express-96 Standard and Deep Well Plates, respectively).
Listeria spp. enrichment (food)
Step 2: Homogenize the food sample.
Step 1: Add 225 mL of BLEB to 25 g
(or 25 mL) of food sample.
Step 2: Twirl the swab for
90±30 seconds.
Step 3: Incubate at 37±1°C under static
conditions for a total incubation time equal
to 28–32 hr. Add Listeria Selective
Enrichment Supplement as directed
by manufacturer.
Step 1a. Prewet the swab with
0.5 mL of D/E Neutralizing Broth.
Step 3: Incubate at 37±1°C under static
conditions for a total incubation time equal
to 24–28 hr. Add Listeria Selective
Enrichment Supplement as directed
by manufacturer.
Using a swab
Listeria spp. enrichment (environmental)
Using a sponge
Step 1c: Add the swab to 10 mL
of BLEB.
Step 1b: Wipe the surface area
to be tested.
Step 2: Homogenize the
environmental sample.
Step 3: Incubate at 37±1°C under static
conditions for a total incubation time equal
to 28–32 hr. Add Listeria Selective
Enrichment Supplement as directed
by manufacturer.
Step 1a. Prewet the sponge with 10 mL
of D/E Neutralizing Broth.
Step 1c: Add the sponge to 100 mL
of BLEB.
Step 1b: Wipe the surface area
to be tested.
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PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Chapter2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria spp.
Sample enrichment 2
Sample enrichment
Enrich of food
sample
1. Add 225 mL of Buffered Listeria Enrichment Broth (BLEB) to 25 g (or 25 mL) of
food sample.
Note: This protocol was validated using Buffered Listeria Enrichment Broth
(VWR # EM1.09628.0500). Other sources of media might provide different results.
2. Homogenize the food sample:
• For solid foods, such as chicken wings, use a filtered Stomacher® bag and
hand massage by squeezing the bag 5–10 times.
• For coarse food types, such as meat and seafood, use a filtered Stomacher®
bag and homogenize for about 1 minute (Stomacher® 400 Laboratory
Blender, or equivalent).
• For soft food types, such as mayonnaise, use a nonfiltered Stomacher® bag
and homogenize for about 1 minute (Stomacher® 400 Laboratory Blender, or
equivalent).
• For liquids or powdered foods, use a nonfiltered Stomacher® bag and hand
massage by squeezing the bag 5–10 times.
3. Incubate at 37±1°C under static conditions for a total incubation time equal to
24–28 hours.
After 4±0.25 hours of incubation or as directed by manufacturer, add Listeria
Selective Enrichment Supplement.
4. Proceed to “DNA isolation (Proteinase K lysis protocol)” on page 14.
Enrich of
environmental
sample using a
swab
1. Collect and enrich the environmental sample.
a. Prewet the swab with 0.5 mL of D/E Neutralizing Broth.
b. Wipe the surface area to be tested.
c. Add the swab to 10 mL of BLEB in a 15-mL conical tube.
2. Twirl the swab for 90±30 seconds.
3. Incubate at 37±1°C under static conditions for a total incubation time equal to
28–32 hours.
After 4±0.25 hours of incubation or as directed by manufacturer, add Listeria
Selective Enrichment Supplement.
4. Proceed to “DNA isolation (Proteinase K lysis protocol)” on page 14.
14 PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Chapter2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria spp.
DNA isolation (Proteinase K lysis protocol)
2
Enrich of
environmental
sample using a
sponge
1. Collect and enrich the environmental sample.
a. Prewet the sponge with 10 mL of D/E Neutralizing Broth.
b. Wipe the surface area to be tested.
c. Add the sponge to 100 mL of BLEB.
2. Homogenize the environmental sample: Use a nonfiltered nonmesh Stomacher®
bag and homogenize for about 1 minute (Stomacher® 400 Laboratory Blender, or
equivalent), or hand squeeze for about 1 minute.
3. Incubate at 37±1°C under static conditions for a total incubation time equal to
28–32 hours.
After 4±0.25 hours of incubation or as directed by manufacturer, add Listeria
Selective Enrichment Supplement.
4. Proceed to “DNA isolation (Proteinase K lysis protocol)” on page 14.
DNA isolation (Proteinase K lysis protocol)
IMPORTANT! Use proper aseptic technique while handling samples to avoid cross-
contamination.
1. Transfer 1 mL of sample to a 1.5-mL microcentrifuge tube.
Note: For environmental samples, squeeze the sponge or twirl the swab 2–3 times
before sampling.
2. Centrifuge the tube at 12,000–16,000 × g for about 3 minutes.
3. Remove and discard the supernatant as quickly as possible to prevent dissipation
of pellet.
Note: If no pellet is visible after centrifugation (for example, as found in filtered
juices), leave ~50 µL of sample in the tube to avoid aspirating the bacterial pellet.
IMPORTANT! For samples that contain a fat layer following centrifugation,
indicated as a distinct top layer, remove the fat layer as follows:
·For liquid fat layer (for example, as found in milk samples): Use a P1000
pipettor to remove fat from the top surface by aspirating in a circular motion
without disturbing the pellet. Continue to collect supernatant from the top
surface until all the supernatant is removed. Discard the supernatant into a
waste container.
or
·For solid fat layer (for example, as found in infant formula samples): Use a
pipettor to gently dislodge the fat layer without disturbing the pellet. Pour off
the supernatant and fat layer using a single quick motion. Aspirate the
supernatant from the top surface using a pipettor until all the supernatant is
removed. Discard the supernatant into a waste container.
4. Add 210 µl of Proteinase K Buffer mix (10 µL PK + 200 µL PK Buffer; see page 11)
to lyse the pellet. Mix well to resuspend the pellet.
15
PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Chapter2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria spp.
DNA isolation (Proteinase K lysis protocol) 2
5. Transfer the sample to a MagMAX™ Express-96 Deep Well Plate (lysis plate).
6. Prepare the elution and wash plates:
a. To prepare the elution plate, add 140 µL of Elution Buffer to those wells of a
MagMAX™ Express-96 Standard Plate (Standard) that correspond to the
lysis plate containing sample.
b. To prepare wash plates 1 and 2, add 300 µL of Wash Buffer to those wells of
two MagMAX™ Express-96 Deep Well Plates (Deep Well) that correspond to
the lysis plate containing sample.
7. Select 44000799DWPrepSEQGP program from the MagMAX™ Express-96
magnetic particle processor. Press Start.
8. Load the plates into the MagMAX™ Express-96 magnetic particle processor
according to the readout. Verify orientation {A1 to A1}.
a. Tip combs — In Standard plate; press Start.
b. Elution plate (140 µL of Elution Buffer) — In Standard plate; press Start.
c. Wash plate 2 (300 µL of Wash Buffer) — In Deep Well plate; press Start.
d. Wash plate 1 (300 µL of Wash Buffer) — In Deep Well plate; press Start.
e. Lysis plate (sample in PK Buffer mix) — In Deep Well plate; press Start.
9. After 20 minutes, the MagMAX™ Express-96 magnetic particle processor will
prompt you to dispense the Lysis Buffer. To dispense the Lysis Buffer:
a. Add 300 µL of Lysis Buffer to each well of the lysis plate containing sample.
b. Load the plate back into the instrument. Press Start.
10. After 18 minutes, the instrument will prompt you to dispense the Binding Mix.
a. Incubate the Magnetic Particles tube at 37±1°C for about 10 minutes, and
then vortex for at least 10 seconds until resuspension is complete.
IMPORTANT! White precipitate can form in the Magnetic Particles during
storage at 5±3°C. Incubate the Magnetic Particles tube at 37±1°C for about
10 minutes prior to use to ensure that no precipitation is present in the
Magnetic Particles.
b. Add 30 µL of Magnetic Particles to each well of the lysis plate containing
sample.
c. Add 300 µL of Binding Solution to each well of the lysis plate containing
sample.
d. Load the plate into the instrument. Press Start.
16 PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Chapter2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria spp.
DNA isolation (Proteinase K lysis protocol)
2
11. When sample preparation is complete, the message “Enjoy your DNA” is
displayed on the screen. Remove the elution plate. Proceed with PCR, or seal the
plate and store below –18°C.
IMPORTANT! For PCR, add 30 µL of eluate to the lyophilized assay. For more
information, refer to the MicroSEQ® Listeria spp. Detection Kit User Guide (Pub.
no. 4426510).
IMPORTANT! If oil droplets are visible as a top layer in the elution plate samples,
then collect eluate from the center of the well (below top layer; see figure below
for instructions on pipetting eluate from food samples with high fat or oil
content).
IMPORTANT! If the elution plate contains magnetic beads, then place the elution
plate on a 96-well magnetic ring stand for at least 1 minute before collecting eluate
for PCR.
IMPORTANT! If the elution plate contains particulate residue from food sample
that does not get removed using the 96-well magnetic ring stand, then centrifuge
the elution plate at about 4000 × g for about 30 seconds to pellet the particulate
residue. Avoid particulate residue when collecting eluate for PCR.
Avoid top layer containing fat/oil
Collect sample for PCR from below fat/oil layer
Avoid residual magnetic particles (if any)
For food samples with high fat or oil content,
an oil/fat layer can form over the DNA sample
in the elution plate. Avoid the top layer and
collect the sample for PCR from below, while
avoiding any residual magnetic particles
(if any) found at the bottom of the plate.
To pipette eluate from foods with high fat or oil content:
17
PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Chapter2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria spp.
Troubleshooting 2
Troubleshooting
Observation Possible cause Recommended action
Inhibition of PCR,
indicated by non-
detection of IPC reaction
Magnetic particles were in the
elution plate.
Avoid disturbing the magnetic particles during transfer
of eluted DNA to the lyophilized assay.
Optional:
Spin the plate at about 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 particulate
residue from food sample.
Avoid residue during transfer of eluted DNA to
lyophilized assay.
Optional:
Spin the plate at about 4000×g for about 30 seconds to
pellet the food residue to the bottom of the plate.
Removal of sample supernatant
was incomplete before addition of
Lysis Buffer.
Ensure maximal removal of the supernatant without
disturbing the bacterial pellet.
The bacterial pellet is
difficult to avoid during
removal of supernatant
The sample was left unattended
before aspirating off the
supernatant, causing dissipation of
the bacterial pellet
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.
The bacterial pellet is
difficult to resuspend
Pellet is too hard. Ensure maximum resuspension of the pellet in the
Lysis Buffer or Proteinase K Buffer before proceeding.
Transfer the entire contents, including the incompletely
resuspended pellet (if any) to the Lysis Plate.
Sample produces a large
pellet upon initial
centrifugation step
(step 2 on page 14:
12,000–16,000×g for
about 3 minutes)
Sample is chunky or has high
debris (for example, chocolate, dry
pet food, or peanut butter sample).
Perform the following PrepSEQ® Preclarification
Protocol:
1. Transfer a fresh 1-mL sample into a 1.5-mL
microcentrifuge tube.
2. Centrifuge the tube containing your sample at about
4000×g for about 1minute.
3. Transfer supernatant to a new 1.5-mL
microcentrifuge tube without disturbing the pellet.
Discard pellet.
4. Centrifuge the tube containing the supernatant at
12,000–16,000×g for about 3 minutes.
5. Proceed to step 3 on page 14.
A
18 PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Supplemental information
Background information and product overview
Description of
target
microorganisms
The genus Listeria is composed of six species; L. monocytogenes, L. innocua,
L. welshimeri, L. seeligeri, L. ivanovii, L. grayi and several recently discovered new
species, given the provisional names L. marthii, L. rocourti, L. fleischmannii, and
L. weihenstephanensis. L. monocytogenes is of clinical relevance for humans because it is
the causative agent for Listeriosis. Listeria monocytogenes poses a severe risk to
pregnant women, potentially causing spontaneous abortion or stillbirth. Normally, L.
monocytogenes is transferred to humans through raw milk, soft-ripened cheeses, raw
vegetables, poultry, raw meats, and raw or smoked fish. L. monocytogenes grows at
temperatures as low as 3°C, allowing it to multiply in refrigerated foods.
Kit sensitivity The sensitivity of the assay in culture samples depends on the quality of the sample
preparation method that is used. The AOAC® Performance Tested MethodsSM workflow
described in this user guide allows you to detect 1 to 3 colony-forming units (CFU)
from 25 grams of food.
Refer to Workflows for Detection of Listeria in Food and Environmental Samples to choose
the appropriate user guide for your laboratory (Pub. no. MAN0009418, available at
www.lifetechnologies.com/foodsafety).
Operating
conditions
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°C decreasing linearly
to 50% relative humidity at 40°C
19
PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
Appendix A Supplemental information
AOAC® Performance Tested MethodsSM Certification A
AOAC® Performance Tested MethodsSM Certification
Visit www.lifetechnologies.com/foodsafety for a list of workflows for the detection of
L. spp.
Workflow The PrepSEQ® kits and the MicroSEQ® Listeria spp. Detection Kit earned the
Performance Tested MethodsSM Certification from the AOAC® Research Institute. The
validated workflow includes:
• Enrichment media: Buffered Listeria Enrichment Buffer (BLEB)
• Sample preparation kit options:
–PrepSEQ
® Nucleic Acid Extraction Kit
–PrepSEQ
® Rapid Spin Sample Preparation Kits
•MicroSEQ
® Listeria spp. Detection Kit
• Applied Biosystems® 7500 Fast Real-Time PCR System
• RapidFinder™ Express Software
• Confirmation testing of positive samples
In the context of AOAC® Validation, when BLEB is used for enrichment media, as
shown in the AOAC®-validated workflow, you can refer to the USFDA Bacteriological
Analytical Manual (BAM), Chapter 10; see www.fda.gov/Food/ScienceResearch/
LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm and scroll to
Listeria monocytogenes.
Matrices The workflow was certified for use with the following matrices:
Confirmation of
results
In terms of AOAC® validation, enriched cultures with positive PCR results were tested
further by cultural confirmation following ISO 11290–1.
Reference method Matrix
ISO 11290–1:1996 with
Amendment 1:2004
Foods: pasteurized whole cow's milk, dry infant formula,
hot dogs, roast beef, lox
Environmental surfaces: stainless steel, plastic cutting
board, ceramic tile, rubber sheets and concrete sealed
with Seal Hard® sealant
B
20 PrepSEQ® Nucleic Acid Extraction Kit for Food and Environmental Testing User Guide: Listeria spp.
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.
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