RapidFinder Campylobacter Multiplex Assay Beads€¦ · The RapidFinder™ Campylobacter Multiplex...

USER GUIDE

For testing of Food and Environmental samples only.

RapidFinder™ Campylobacter Multiplex Assay BeadsReal-time PCR detection of C. jejuni, C. coli, and/or C. lari from food samples

Catalog Number 4485027

Publication Number 4466312 Revision B

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

DISCLAIMER

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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.

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.

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Life Technologies is a Thermo Fisher Scientific brand. © 2014 Thermo Fisher Scientific Inc. All rights reserved.

Contents

3RapidFinder™ Campylobacter Multiplex Assay Beads User Guide

About this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

■ RapidFinder™ Campylobacter Multiplex Assay Beads . . . . . . . . . . . . . . . . 7

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Kit components and storage conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Materials not included in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Isolate DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Prepare for PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Create a run file document in the SDS software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Prepare the assay beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Prepare samples and controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Prepare tubes for the 7500 Fast System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Start instrument run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Run the 8-tube reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

View results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15General process using SDS software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Resources for viewing results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

■ APPENDIX A Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . 19

Description of target microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Kit sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Kit specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Audience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Operational conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Good PCR practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Prevent contamination and nonspecific amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

■ APPENDIX B Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

4 RapidFinder™ Campylobacter Multiplex Assay Beads User Guide

Contents

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Obtaining information from the Help system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Obtaining Certificates of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Food safety support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26


Contents


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

B January 2014 • Update number formatting for temperature, time, and centrifugal speeds.• Update product name from MicroSEQ® Campylobacter jejuni/coli/lari Detection

Kit and update catalog number to reflect the change from a custom assay to a catalog assay.

• Updated to a new Life Technologies template with associated updates to the limited license information, warranty information, trademark statement, and safety statements.

A May 2011 New user guide.


RapidFinder™ Campylobacter Multiplex Assay BeadsProduct description

RapidFinder™ Campylobacter Multiplex AssayBeads

Product description

The RapidFinder™ Campylobacter Multiplex Assay Beads uses a simple, reliable, and rapid assay to detect Campylobacter jejuni/coli/lari from enriched food samples and poultry carcass rinses. The assay uses real-time polymerase chain reaction (PCR) and TaqMan® probes to amplify and detect unique microorganism-specific DNA target sequences.

The lyophilized assay beads provided with the kit contain all of the reagents (except for template DNA) required for detection of C. jejuni, C. coli, and/or C. lari, as well as an Internal Positive Control (IPC) to identify possible PCR inhibition. This IPC also eliminates the need for a positive control, which reduces the risk of cross-contamination in unknown samples. User-supplied positive control(s) are optional.

This guide provides instructions for use on the Applied Biosystems® 7500 Fast Real-Time PCR System. For use on other real-time PCR instruments, consult your instrument user guide.

Note: We recommend that the user perform validation with their unique sample matrices/types to determine appropriate analysis settings (ISO 22174, 2005). Life Technologies offers fee-based method validation and verification services; contact [email protected] for more information.

Kit components and storage conditions

The RapidFinder™ Campylobacter Multiplex Assay Beads (Cat. no. 4485027) contains reagents for 96 assays.

Description Amount Storage Conditions†

† Refer to the product label for expiration date.

Campylobacter jejuni/coli/lari Assay Beads‡, 8-tube strips

‡ Lyophilized assay beads contain Campylobacter jejuni/coli/lari-specific probes and primers, internal positive control (IPC) probe and primers, IPC template, enzyme, and other buffer components.

12 strips (96 caps)

1 rack

2 to 8°C

Protect from light§ and seal pouch tightly.††

§ Excessive exposure to light may affect the fluorescent probes.††Each time you remove 8-tube strips from the pouch, seal the pouch tightly to protect contents from moisture.

MicroAmp® Optical 8-Cap Strips 12 strips (96 caps) Room temperature (23±5°C)


RapidFinder™ Campylobacter Multiplex Assay Beads Materials not included in the kit

Materials not included in the kit

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

Item Source†

† The materials listed here have been validated for use with this kit. Results may vary if substituted products from other vendors are used instead.

Instruments

Applied Biosystems® 7500 Fast Real-Time PCR System Contact your local Life Technologies sales office.

Equipment

Benchtop microcentrifuge MLS

Plate centrifuge MLS

Vortexer MLS

7500 Fast Precision Plate Holder for MicroAmp® Tube Strips (for use with 7500 Fast Real-Time PCR System)

Cat. no. 4403809

MicroAmp® 96-Well Base Cat. no. N8010531

MicroAmp® Cap Installing Tool Cat. no. 4330015

Consumables

Aerosol-resistant pipette tips MLS

Disposable gloves MLS

Pipettors:• Positive-displacement• Air-displacement• Multichannel

MLS

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

MicroAmp® Optical 8-Cap Strips Cat. no. 4323032

Reagents

Nuclease-free Water Cat. no. AM9938


RapidFinder™ Campylobacter Multiplex Assay BeadsWorkflow

Workflow

This guide provides instructions for using the RapidFinder™ Campylobacter Multiplex Assay Beads with the Sequence Detection System (SDS) software on the 7500 Fast Real-Time PCR System.

Isolate DNA

For DNA isolation, we recommend that you use the protocol described in the PrepSEQ® Rapid Spin Sample Preparation Kits: Campylobacter jejuni/coli/lari User Guide (Pub. no. 4466620). This protocol contains procedures for sample enrichment and purification and lysate preparation using a spin-column based procedure. Use the protocols with one of the following kits:

• PrepSEQ® Rapid Spin Sample Preparation Kit• PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean

Prepare for PCR

To prepare for PCR, first create a run file document, then prepare the assay beads and samples.

PrepSEQ® Rapid Spin Sample Preparation Kit†

† Refer to the PrepSEQ® Rapid Spin Sample Preparation Kit: Campylobacter jejuni/coli/lari User Guide (Pub. no. 4466620) for instructions on DNA isolation.

Isolate DNA (see page 9)

Enrich food sample (or poultry carcass rinses)

Purify sample using Rapid Spin column

Prepare sample lysate

RapidFinder™ Campylobacter Multiplex Assay Beads with SDS

Software (covered in this guide)

Prepare for PCR (see page 9)

Create a run file document in the SDS software (see page 10)

Prepare the assay beads (see page 12)

Prepare samples and controls (see page 12)

Prepare tubes for the 7500 Fast System (see page 13)

Start instrument run (see page 14)

Run the 8-tube reactions (see page 14)

View results (see page 15)


RapidFinder™ Campylobacter Multiplex Assay Beads Prepare for PCR

Create a run file document in the SDS software

Note: The NED™ dye is used to detect the internal positive control (IPC); the FAM™, VIC®, and LIZ® dyes are used to detect the targets.

1. Open the SDS software for the 7500 Fast System.

2. Select File New to open the New Document Wizard.

3. In the Define Document section of the New Document Wizard:

a. Select or enter the following information:Assay – Select Absolute Quantification (Standard Curve)Container – Select 96-Well ClearTemplate – Select Blank DocumentRun Mode – Select 7500 FastAll other fields – Enter the requested information

b. Click Next to continue.

Note: For more information on creating a run file document, refer to the documentation that is provided with your instrument.

4. In the Select Detectors section of the New Document Wizard:

a. For each of the following detectors, select the detector, then click Add to add it to the Detectors in Document field:

• Reporter FAM/Quencher none (C. coli)• Reporter VIC/Quencher none (C. jejuni)• Reporter LIZ/Quencher none (C. lari)• Reporter NED/Quencher none (IPC)

b. Click Next to continue.

Note: If a detector is missing from the list, click New Detector and create a new detector for each reporter dye. Select a different color for each reporter dye.

5. In the Set Up Sample plate section of the New Document Wizard:

a. Assign the four detectors (FAM, VIC, LIZ, and NED) to each well that contains sample, then click the Use checkbox next to each detector.

IMPORTANT! Review “Start instrument run” on page 14 to determine the optimal sample layout in order to minimize bending or misaligning the tube strips.

Note: If any of the four detectors are missing from the list of detectors, use the Back tab, follow the directions in step 4 to add the missing detectors to the document.

b. Confirm that each sample well contains four “U” symbols corresponding to the four reporter dyes (FAM™, VIC®, LIZ®, and NED™ dyes).

c. Click Finish to continue.

6. In the Setup tab, enter sample names for each sample by double-clicking the sample well.


RapidFinder™ Campylobacter Multiplex Assay BeadsPrepare for PCR

7. In the Instrument tab, set thermal cycling conditions as indicated in the following table:

For more details, refer to the 7300/7500/7500 Fast Real-Time PCR System Absolute Quantitation Using Standard Curve Getting Started Guide.

8. Set Sample Volume to 30 µL.The following image shows thermal cycling conditions for the 7500 Fast System using SDS software with run mode set to Fast 7500:

Stage Temperature Time

Hold 95°C 2 minutes

Cycle (40 cycles) Denature 95°C 3 seconds

Anneal/extend 60°C 30 seconds


RapidFinder™ Campylobacter Multiplex Assay Beads Prepare for PCR

Prepare the assay beads

1. Open the storage pouch containing the assay beads (cut at the notch in the top 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 based on the number of samples and controls that you plan to run, and place them in a 96-well base. At least one negative control is recommended for the target pathogen. Remove colored caps and discard. Avoid disturbing the beads from the bottom of the tubes.

Note: The assay beads contain all the components necessary for each reaction.

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

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

Prepare samples and controls

1. Thaw all reagents (samples and controls) completely.

2. Before opening thawed sample and control tubes, centrifuge tubes in a microcentrifuge at maximum speed for 1 minute to avoid cross contamination and to separate any particulate material derived from the spin column, which can interfere with amplification. The microbial DNA is in the aqueous phase.

3. Dispense all unknown samples, followed by negative control(s), then by positive control(s) as follows:

a. Using a new pipette tip for each sample or control, add 30 µL of sample or control to an assay bead.The bead dissolves in 1–5 seconds.

b. Gently pipet up and down a few times with the pipette tip at the bottom of the tube to mix. Pipet gently to minimize aerosol formation and cross-contamination. (Alternatively, after the tubes are capped in the final step, vortex the capped tubes until the pellet is dissolved.)

IMPORTANT! Prepare and close all negative-control and unknown sample tubes before pipetting the (optional) positive control.

IMPORTANT! Food samples with high fat or oil content can form a top layer containing fat and debris over the DNA sample. When collecting your sample for PCR, avoid the top layer and bottom pellet by collecting your sample from the clear center section (see the following figure).


RapidFinder™ Campylobacter Multiplex Assay BeadsPrepare for PCR

Note: The PrepSEQ® Rapid Spin Sample Preparation Kits provide ~300 µL of sample. If you use other sample preparation methods, and less than 30 µL of sample is available, adjust the sample or positive control volume with water to bring the total volume to 30 µL.

Prepare tubes for the 7500 Fast System

8-tube strips containing assay beads are compatible with the 7500 Fast System.

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 empty capped 8-tube strips evenly distribute the clamping load that is applied to the sample tube strips during processing, thereby minimizing the risk of collapsing any tubes. Add empty tubes as needed to create a complete 8-tube strip and cap with an intact 8-cap strip.

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 and the MicroAmp® Cap Installing Tool 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. Mark or label one end of the strip cap (but not over a cap) so that you will remember the orientation when transferring the tubes to the instrument tray.

4. Make sure that the reactions are thoroughly mixed. If reactions were not previously mixed during the pipetting step, place the assay tubes in a 96-well base and use a vortexer to mix.

5. Make sure that the reagents are at the bottom of the tubes. If available, centrifuge the tube contents at 1000–2000 × g for about 30 seconds using a centrifuge with a plate adapter.

Avoid top layer containing fat/oil

Collect sample for PCR from below fat/oil layer

Avoid particulate material from column

To pipet eluate from foods with high fat or oil content:


RapidFinder™ Campylobacter Multiplex Assay Beads Start instrument run

Start instrument run

This section describes the procedure for running the 8-tube reactions on the Applied Biosystems® 7500 Fast System using SDS software.

We recommend using the 7500 Fast Precision Plate Holder for MicroAmp® Tube Strips.

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

1. If you are not using columns 1 (leftmost) and 12 (rightmost) for samples, insert two empty, capped MicroAmp® Fast 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.

2. 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: You can run a minimum of two and a maximum of twelve 8-tube strips at one time.

IMPORTANT! Always use a total of 8 tubes per column. Add new, empty, capped MicroAmp® Fast tubes to a column as needed.


RapidFinder™ Campylobacter Multiplex Assay BeadsView results

3. Open the run file document that corresponds to the reaction plate that you created in “Create a run file document in the SDS software” on page 10, then save the run file.

4. In the Instrument tab, select Start to begin the run.

Note: You must save the run file in order for the run to start. If you did not save the run file before selecting Start, a message appears prompting you to save the run file.

IMPORTANT! To avoid false positives due to amplified material in your work area, do not open tubes after amplification.

View results

General process using SDS software

The general process for viewing results from RapidFinder™ Campylobacter Multiplex Assay Beads is as follows:

1. View the amplification plots for all reactions.

2. Set the baseline and threshold values:

a. For the baseline, use the default settings (start cycle 3 to end cycle 15).

b. Set the threshold value to 0.2.

3. For each sample, determine the CT value for each dye, then determine if the sample is positive or negative for each species based on the table below.

Dye signal CT†

† Threshold cycle (CT) is the PCR cycle number at which the fluorescence meets the threshold in the amplification plot.

Result

FAM™ dye signal (C. coli)≤37 Positive‡ for C. coli

‡ Result is positive whether or not IPC is detected.

>37 Negative§ for C. coli

§ See “Troubleshooting” on page 16, if the IPC is not detected.

VIC® dye signal (C. jejuni)≤36 Positive‡ for C. jejuni

>36 Negative§ for C. jejuni

LIZ® dye signal (C. lari)≤37 Positive‡ for C. lari

>37 Negative§ for C. lari

NED™ dye signal (IPC)

IPC signal detected Positive for IPC

“Undetermined” in SDS software

Negative for IPC; if no IPC is detected and there are no positive target-specific results, the result is inconclusive.


RapidFinder™ Campylobacter Multiplex Assay Beads Troubleshooting

Resources for viewing results

For more information about analyzing your data, refer to the appropriate instrument user guide.

We recommend that you use different methods to screen samples and confirm results. When you use the RapidFinder™ Campylobacter Multiplex Assay Beads to screen samples, it is recommended that you confirm the results using culture and biochemical methods. FSIS (USDA) MLG 41.02 and ISO 10272-1:2006 (see “References” on page 24) are approved protocols for confirmation.

Troubleshooting

Observation Possible cause Recommended action

No IPC signal and no target-specific signal is detected in unknown wells. (Note: When there is target-specific signal in the absence of IPC signal for an unknown sample, the result is considered positive and no troubleshooting is required.)

Inhibition of PCR occurred. Add 5 µL of sample and 25 µL of water to the lyophilized assay to overcome inhibition. Alternatively, repeat sample preparation to obtain a new sample for PCR.

Note: For more information, refer to the “Troubleshooting” section of the PrepSEQ® Rapid Spin Sample Preparation Kit User Guide: Campylobacter jejuni/coli/lari (Pub. no. 4466620).

Inhibition of PCR, indicated by non-detection of IPC reaction.

The supernatant was not sufficiently removed before Lysis Buffer was added.

Add 5 µL of sample and 25 µL of water to the lyophilized assay to overcome inhibition.

Filtrate from the spin column is present in the PCR reaction.

Centrifuge the sample to separate the filter particulates before adding sample to the PCR reaction. Pipet clear sample from the center section of the tube to avoid the particulate material at the bottom of the tube.

The sample contains excess fat that was not removed during aspiration of the supernatant.

Use the PrepSEQ® Rapid Spin Sample Preparation Kit – Extra Clean when processing a matrix with high fat content.

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 pipet positive control into all positive-control wells.

No IPC is detected, but target-specific 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 Life Technologies Food Safety Technical Support.


RapidFinder™ Campylobacter Multiplex Assay BeadsTroubleshooting

In negative-control wells, a target-specific 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

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.

Replicate results for this sample are inconsistent.

All replicate wells for a sample do not have the same result.

If more than two replicates yield the same result (for example, 2 of 3 replicates are negative, but 1 replicate is positive), refer to your laboratory protocol to determine whether to repeat the assay using fresh samples and reagents.

If only 2 replicates were run and the results are not consistent, repeat the assay using fresh samples and reagents.

Very strong signal (low CT in no template control), indicating a false positive.

Amplicon contamination was introduced, either:• Into the PCR clean area from

post-amplification reaction tubes that were either opened in the clean area or brought into the PCR clean area from contaminated gloves or solutions

• Into the real-time PCR instrument from crushed and broken PCR reaction tubes

To investigate possible amplicon contamination:

1. Prepare negative control samples (at least one 8-tube strip) using the lyophilized assay beads (from the RapidFinder™ Campylobacter Multiplex Assay Beads):a. Add 30 µL of nuclease-free water to 4 of

the assay beads.b. To the remaining 4 assay beads, add:

• 29 µL of nuclease-free water• 1 µL of 1 U/µL Uracil DNA

Glycosylase (Cat. no. 18054-015).

2. Run samples on the 7500 Fast instrument:• In the SDS software, select the Fast

7500 Run Mode. • In the Instrument tab, under stage 1,

select Add Step to stage 1 of the PCR cycle that consists of 10 minutes at 37°C, then extend the 95°C step from 20 seconds to 10 minutes.

Results that show target-specific signal in the –UNG samples and no target-specific signal in +UNG samples are evidence of amplicon contamination.

If the instrument block was contaminated, consult the 7300/7500/7500 Fast Real-Time PCR System Absolute Quantitation Using Standard Curve Getting Started Guide (Pub. no. 4347825) and/or contact a service representative to clean the instrument.

Observation Possible cause Recommended action


RapidFinder™ Campylobacter Multiplex Assay Beads Troubleshooting


A Supplemental information

Description of target microorganisms

Campylobacter jejuni/coli/lari are major foodborne pathogens that cause diarrhea, periodontitis, and dysentery syndrome in humans. Campylobacter jejuni is the leading cause of bacterial diarrheal illness in the United States. It causes more disease than Salmonella spp. Outbreaks of Campylobacteriosis have been associated with contaminated food supplies such as chicken (20–100% of retail chickens are contaminated), raw milk, and leafy greens.

Kit sensitivity

The limit of detection using this kit is 103 colony-forming units (cfu)/mL for enriched food matrices, or enriched or direct poultry carcass rinses.

The sensitivity of the assay in real culture samples depends on the quality of the sample preparation method that is used. The PrepSEQ® Rapid Spin Sample Preparation Kit: Campylobacter jejuni/coli/lari User Guide (Pub. no. 446620) contains sample preparation procedures that allow you to detect 1–3 cfu in 25 grams of food or 25 mL of enriched poultry carcass rinses, or 100–1000 cfus in 25 mL of direct poultry carcass rinses.

Kit specificity

The RapidFinder™ Campylobacter Multiplex Assay Beads specifically detects the Campylobacter jejuni/coli/lari serotypes.

Audience

This document is intended for investigators who need to test for Campylobacter jejuni/coli/lari in food samples, or enriched or direct poultry carcass rinses.


Appendix A Supplemental informationOperational conditionsA

Operational conditions

The Applied Biosystems® 7500 Fast Real-Time PCR System is for indoor use only and for altitudes not exceeding 2000 m (6500 ft.) above sea level.

Good PCR practices

Prevent contamination and nonspecific amplification

PCR assays require special laboratory practices to avoid false positive amplifications. The high throughput and repetition of these assays can lead to amplification of one DNA molecule.

PCR good laboratory practices

When preparing samples for PCR amplification:

• Wear clean gloves and a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation).

• Change gloves whenever you suspect that they are contaminated.• Maintain separate areas and dedicated equipment and supplies for:

– Sample preparation– PCR setup– PCR amplification – Analysis of PCR products

• Never bring amplified PCR products into the PCR setup area.• Open and close all sample tubes carefully. Try not to splash or spray PCR

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

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.

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


B 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. 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.


Appendix B SafetyChemical safetyB

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 SDS for specific precautions and instructions:• Read and understand the Safety Data Sheets (SDSs) provided by the

chemical manufacturer before you store, handle, or work with any 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.

WARNING! HAZARDOUS WASTE (from instruments). Waste produced by the instrument is potentially hazardous. Follow the guidelines noted in the preceding General Chemical Handling warning.

WARNING! 4L Reagent and Waste Bottle Safety. Four-liter reagent and waste bottles can crack and leak. Each 4-liter bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position.


Appendix B SafetyBiological hazard safety B

Biological hazard safety

WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surface may be considered a biohazard. Use appropriate decontamination methods when working with biohazards.

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


References

ISO. 2005. Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the detection of food-borne pathogens – General requirements and definitions. Reference number 22174:2005.

ISO. 2006. Microbiology of food and animal feeding stuffs — Horizontal method for detection and enumeration of Campylobacter spp. Reference number 10272-1:2006(E).

Food Safety and Inspection Service (USDA). 2013. Isolation, identification and enumeration of Campylobacter jejuni/coli/lari from poultry rinse, sponge and raw product samples. MLG 41.02. Microbiology Laboratory Guidebook.


Documentation and support

Related documentation

Portable document format (PDF) versions of this guide and the following related documents are available online at www.lifetechnologies.com:

Note: To open the user documentation available online, use the Adobe® Reader® software available from www.adobe.com

Note: For additional documentation, see “Obtaining support” on page 26.

Obtaining information from the Help system

The SDS software has a Help system that describes how to use each feature of the user interface. Access the Help system by doing one of the following:

• Click in the toolbar of the SDS software window.• Select Help Contents and Index.• Press F1.

You can use the Help system to find topics of interest by:

• Reviewing the table of contents• Searching for a specific topic• Searching an alphabetized index

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.

Document Pub. no. Description

PrepSEQ® Rapid Spin Sample Preparation Kits User Guide

4466620 Describes procedures for setting up the SDS software, running PCR, and analyzing results for Campylobacter jejuni/coli/lari detection.

7300/7500/7500 Fast Real-Time PCR System Absolute Quantitation Using Standard Curve Getting Started Guide

4347825 Provides procedures for designing and performing absolute quantitation experiments using the 7500 Fast Real-Time PCR System.

SDS Software v1.4 Help N/A Describes the SDS Software v1.4 and provides procedures for common tasks.

http://www.lifetechnologies.com/support


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http://www.appliedbiosystems.com/support



http://www.adobe.com

http://www.adobe.com


Documentation and support Obtaining Certificates of Analysis

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

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 (In North America): 1-800-500-6885

Phone number (Outside of North America): Go to www.lifetechnologies.com/contactus.html and select the appropriate country from the drop-down menu.

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.







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