Thermo Fisher Scientific PrepSEQ Nucleic Acid Extraction Kit: Listeria monocytogenes User guide

Brand: Thermo Fisher Scientific

Model: PrepSEQ Nucleic Acid Extraction Kit: Listeria monocytogenes

Type: User guide

USER GUIDE

For testing of Food and Environmental samples only.

PrepSEQ® Nucleic Acid Extraction Kit:

Listeriamonocytogenes

Preparation of MicroSEQ® PCR-ready DNA from food

samples

For use with: MagMAX™ Express-96 Deep Well Magnetic Particle Processor

Catalog Numbers 4428176, 4480466

Publication Number 4405966

Revision D

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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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

outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

TRADEMARKS

The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. AOAC is a registered

trademark and Performance Tested Methods is a service mark of AOAC International. Stomacher is a registered trademark of Seward Limited, UK. Whirl-

Pak is a registered trademark of Aristotle Corporation.

Contents

PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

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 monocytogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Kit workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Enrichment of food sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

DNA isolation (Proteinase K lysis protocol) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

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 Testing User Guide: Listeria monocytogenes

Contents

Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Obtaining Certificates of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Food safety support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

Contents

6PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

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

D November 2013 • Added certification details for AOAC® Performance Tested MethodsSM

certification.

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

AOAC® certification.

• Added additional troubleshooting tip.

• Updated to a Life Technologies template with associated updates to the

limited license information, warranty information, trademark statement, and

safety statements.

C June 2010 Baseline for revision history.

PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

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 samples to test for

Listeria monocytogenes. The kit procedure involves:

• Enrichment of food samples for Listeria monocytogenes

• Nucleic acid extraction

To extract DNA from Listeria monocytogenes in food samples, we recommend the

Proteinase K lysis protocol that is described on page 13.

For the isolation and purification of DNA from enriched food samples, a 1-mL sample

volume is recommended. The recommended elution volume is 140 µL.

See Appendix A, “Supplemental information” for detailed information about AOAC®

certification.

The PrepSEQ® Rapid Spin Sample Preparation 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 monocytogenes.

8PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

Chapter1 Product information

Kit contents

Table1 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,

all materials are available from Life Technologies. MLS: Major laboratory supplier.

Item Cat. no.4480466

(100 reactions)

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

PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

Chapter1 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

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

Microcentrifuge tubes, PCR clean, 1.5-mL MLS

Homogenizer bags appropriate for your sample:

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

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 Testing User Guide: Listeria monocytogenes

Chapter1 Product information

Materials not included in the kit

PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

PrepSEQ® Nucleic Acid Extraction Kit:

Listeria monocytogenes

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

A sample preparation workflow based on the Proteinase K lysis protocol is shown

below for procedures starting on page 13.

12 PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

Chapter2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria monocytogenes

Kit workflow

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 all 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).

L. monocytogenes 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 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.

PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

Chapter2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria monocytogenes

Enrichment of food sample 2

Enrichment of food sample

1. Add 225 mL of Buffered Listeria Enrichment Broth (BLEB) to 25 g (or 25 mL) of

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 sample:

• For coarse food types, such as meat, poultry, 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 or soft cheese, 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 13.

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.

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.

14 PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

Chapter2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria monocytogenes

DNA isolation (Proteinase K lysis protocol)

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.

·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.

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 all of the processing 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 into the instrument. Press Start.

PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

Chapter2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria monocytogenes

DNA isolation (Proteinase K lysis protocol) 2

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.

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

information, refer to the MicroSEQ® Listeria monocytogenes Detection Kit User

Guide (Pub. no. 4405962).

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.

16 PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

Chapter2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria monocytogenes

DNA isolation (Proteinase K lysis protocol)

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:

PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

Chapter2 PrepSEQ® Nucleic Acid Extraction Kit: Listeria monocytogenes

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.

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 13:

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 1minute.

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 13.

18 PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

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

PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

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. monocytogenes.

Workflow The PrepSEQ® kits and the MicroSEQ® Listeria monocytogenes 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 monocytogenes 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,

ice cream, roast beef, cured bacon, lox, lettuce, salad

dressing, mayonnaise

20 PrepSEQ® Nucleic Acid Extraction Kit for Food Testing User Guide: Listeria monocytogenes

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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Thermo Fisher Scientific PrepSEQ Nucleic Acid Extraction Kit: Listeria monocytogenes User guide

Thermo Fisher Scientific PrepSEQ Nucleic Acid Extraction Kit: Listeria monocytogenes User guide

Thermo Fisher Scientific PrepSEQ Nucleic Acid Extraction Kit: Listeria monocytogenes User guide

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