Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit

Transcription

1 USER GUIDE Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit Catalog Numbers APS500P (Pathatrix kit), (MicroSEQ kit) Publication Number MAN Revision A.0 For testing of Food and Environmental samples only.

2 The information in this guide is subject to change without notice. DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. LIMITED USE LABEL LICENSE No. 492: Environmental Testing, Quality Control/Quality Assurance Testing, Food and Agricultural Testing Notice to Purchaser: The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product (a) to perform internal research for the sole benefit of the purchaser; and (b) for environmental testing, quality control/quality assurance testing, food and agricultural testing, including reporting results of purchaser's activities in environmental testing, quality control/quality assurance testing, food and agricultural testing for a fee or other commercial consideration. No other right is hereby granted expressly, by implication, or by estoppel. This product is for environmental testing, quality control/ quality assurance testing, food and agricultural testing and research purposes only. The purchase of this product does not grant the purchaser any additional rights, including (without limitation) the right to transfer or resell the product in any form or the right to use the product as a therapeutic agent or diagnostics test component. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 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. BBL is a trademark of Becton, Dickinson and Company. CHROMagar is a trademark of Alain Rambach. Oxoid and Brilliance are trademarks of Oxoid Limited. Stomacher is a registered trademark of Seward Limited, UK. Whirl- Pak is a registered trademark of Aristotle Corporation Life Technologies Corporation. All rights reserved.

3 Contents About this guide Revision history CHAPTER 1 Product information Overview Kit contents Sample preparation kit Real-time PCR kit Required materials not included in the kits CHAPTER 2 Methods Workflow Procedural guidelines Sample preparation Sample pooling Samples and consumable loading Sample unloading DNA sample preparation for real-time PCR Real-time PCR setup Real-time PCR detection Retesting of individual samples after a positive pooled sample Interpretation of positive results by plating and biochemical assays Test result interpretation and classification Result warnings and recommendations for retesting APPENDIX A Alternative enrichment methods Prepare potentially acidic/alkaline samples for enrichment Dairy samples Chocolate and cocoa-based samples Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 3

4 Contents APPENDIX B Troubleshooting General troubleshooting Investigating warning results or failed runs in the SDS Software Interpretation of amplification plot for samples with a Result Warning APPENDIX C Supplemental information Product information Description of target microorganism Audience Applicability Sampling protocol Kit sensitivity Operating conditions AOAC Performance Tested Methods SM Certification Good PCR practices: prevent contamination and nonspecific amplification PCR good laboratory practices Plate layout suggestions APPENDIX D Safety Chemical safety Biological hazard safety References Documentation and support Obtaining SDSs Obtaining support Obtaining Certificates of Analysis Food safety support Limited product warranty Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

5 Contents Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 5

6 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 A.0 December 2013 Added logos and details for AOAC Performance Tested Methods SM certification. Added general troubleshooting tips Used updated numbering system for document revision labeling Used updated user guide template with associated updates to the general document organization, limited license information, trademark statement, safety statements, and support information. 1.0 February 2013 New user guide. 6 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

7 1 Product information Ensure that your instruments are properly installed and calibrated. For calibration information, see the documentation that is provided with your instruments. Overview The Pathatrix 10-Pooling Salmonella spp. Kit is a sample preparation method for presence/absence testing based on the detection of as few as 1 10 cfu (colony forming units) per g of food sample. Using the Pathatrix 10-Pooling Salmonella spp. Kit linked to the MicroSEQ Salmonella spp. Detection Kit, presumptive results can be obtained, prior to confirmation, in as little as 20 hours. A presumptive positive isolate should be subsequently confirmed by the use of subculture, as well as appropriate biochemical and serological tests as required. Once confirmed, the results are reported as: Salmonella spp. Detected in g (sample matrices) Salmonella spp. Not detected in g (sample matrices) This guide describes an AOAC Performance Tested Methods SM workflow for detection of Salmonella spp. in food samples (see AOAC Performance Tested Methods SM Certification on page 35 for details). Visit for a list of workflows for detection of Salmonella spp. Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 7

8 1 Chapter 1 Product information Kit contents Kit contents Note: Parts may ship separately depending on configuration and storage conditions. Sample preparation kit The Pathatrix 10-Pooling Salmonella spp. Kit (Cat. no. APS500P) contains enough consumable components and Pathatrix beads to process 500 samples (50 Cartridge runs). Item Quantity or volume Storage Presterilized Sample and Elution Vessel Packs Presterilized Capture Phase Packs Presterilized Flat Cap Lids Anti-Salmonella spp. Antibody-Coated Paramagnetic Beads 50 each Room temperature (23±5 C) 2.5 ml (50 tests) 5±3 C Refer to the product label for expiration date. IMPORTANT! Never freeze the Pathatrix beads. Beads that have been subjected to freezing temperatures may be rendered inactive. Real-time PCR kit Item MicroSEQ Salmonella spp. Detection Kit Pathogen Detection Negative Control MicroSEQ Salmonella spp. Detection Kit (Cat. no ) contains reagents for 96 reactions. Description Amount Cap color Storage Salmonella spp. Assay Beads, 8-tube strips 12 strips (96 caps) 1 rack Green (rack) 5±3 C protect from light seal pouch tightly MicroAmp Optical 8-Cap Strips 12 strips (96 caps) N/A Room temperature (23±5 C) Pathogen Detection Negative Control 1.5 ml Red 5±3 C Refer to the product label for expiration date. Lyophilized assay beads contain Salmonella spp.-specific probe and primers, internal positive control (IPC) probe, primers, IPC template, enzyme and other buffer components 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. 8 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

9 Chapter 1 Product information Required materials not included in the kits 1 Required materials not included in the kits Unless otherwise indicated, all materials are available from Life Technologies. MLS: major laboratory supplier. Item Source Equipment Incubator, 37±1 C Heating block, 97±2 C Homogenizer (e.g., Stomacher 400 Laboratory Blender) Pathatrix Auto Instrument, including Sample Vessel Holder and Elution Vessel Holder Magnetic-particle concentrator, for example: DynaMag -2 Magnet (for use with microcentrifuge tubes) Magnetic rack (for use with 96-well plate, such as Pathatrix Magnetic Plate or Magnetic Stand-96) MLS MLS Seward #0400/001/AJ, or equivalent Cat. no. PATHATRIXAUTO Cat. no D MicroAmp 96-Well Base Cat. no. N MicroAmp Cap Installing Tool Cat. no Fast Precision Plate Holder for MicroAmp Tube Strips Cat. no Food Pathogen Detection System Package: Applied Biosystems 7500 Fast Real-Time PCR System With PC Tower and RapidFinder Express Software or With Notebook Computer and RapidFinder Express Software (Optional) Pathatrix Cartridge Rack; holds 5 Cartridges Consumables Optional for environmental samples: Swabs or sponges For enrichment: Sterile bags with internal strainers [for most food samples; e.g., Stomacher or Whirl-Pak bags] Sterile bags [for liquid samples; e.g., Stomacher or Whirl-Pak bags] Sterile 15-mL tubes [optional, for use with swabs] Optional, for high-particulate or high-fat-content samples in sterile bags: Foam filters 10-Pool Kit Straws (254 mm) and Syringes (10 ml) Cat. no. MAGNETICPLATE or AM10027 Contact your local Life Technologies representative. ACARTRACK MLS MLS (e.g., Seward product code BA6041/STR, Nasco # B01348WA, or equivalent) MLS (e.g., Seward product code BA6041, Nasco # B01196WA, or equivalent) MLS Cat. no. PFF Cat. no. POOL1010MLN Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 9

10 1 Chapter 1 Product information Required materials not included in the kits Item Source For DNA sample preparation: Sterile 1.5-mL microcentrifuge tubes MicroAmp Fast optical 96-Well Reaction Plate with Barcode, 0.1 ml (optional, for processing large numbers of samples) MicroAmp Clear Adhesive Film (optional, for processing large numbers of samples) Sterile, disposable 10-µL loops Media Optional, for prewetting swabs or sponges: Dey-Engley Neutralizing Broth For enrichment: Buffered peptone water (for most samples) UHT skim milk or nonfat dried milk, reconstituted (for chocolate or cocoa-based samples) Brilliant green (CAS ) (for use with dairy, chocolate, and cocoa-based samples) Selective agar (for confirmation testing) XLD agar Second selective agar, such as: Brilliant Green Agar (modified) Salmonella-specific Chromogenic agar, such as: Oxoid Brilliance Salmonella Agar Biokar Diagnostics IRIS Salmonella Agar or BBL CHROMagar Salmonella MLS Cat. no Cat. no MLS BD product code , or equivalent Oxoid product code CM0509, or equivalent Food retail store MLS Oxoid product code CM0469 Oxoid product code CM0329, or equivalent Oxoid product code CM1092 Biokar Diagnostics product code BM160 or BD product number Reagents PBS, 10X, ph 7.4 Cat. nos. AM9624 or AM9625 (Dilute 1:10 in sterile water prior to use.) Nuclease-free water Cat. no. AM9938 Lysis Buffer, FS Cat. no See page 12 and Appendix A on page 25 for recommendations about enrichment media choice. Media is supplied by several manufacturers [e.g., Oxoid, BD, Difco, and Merck] in a dehydrated form and should be prepared according to the manufacturer's instructions. 10 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

11 2 Methods Workflow Sample preparation (page 12) Sample pooling (page 13) Samples and consumable loading (page 14) Sample unloading (page 16) DNA sample preparation for real-time PCR (page 17) Real-time PCR setup (page 19) Real-time PCR detection (page 19) Retesting of individual samples after a positive pooled sample (page 22) Interpretation of positive results by plating and biochemical assays (page 22) Test result interpretation and classification (page 23) Procedural guidelines Use aseptic technique and good laboratory practices at all times. A facemask should be worn when weighing out powders. Care must be taken when boiling agar prior to autoclaving, and heat-resistant gloves should be worn when handling hot flasks of liquid. Take care when handling plates or vessels that contain microorganisms. Avoid generating aerosols, as pathogenic organisms may be present. Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 11

12 2 Chapter 2 Methods Sample preparation Used or unused reagents, used media, sample enrichments, as well as other contaminated disposable materials should be disposed of following procedures for infectious or potentially infectious products. Sample enrichments might be contaminated with pathogenic organisms infectious to humans, so all waste must be treated as biohazardous and handled and disposed using safe laboratory practices, in accordance and compliance with all appropriate regulations. Sample preparation Note: See AOAC Performance Tested MethodsSM Certification on page 35 for details about matrices used in the AOAC validation study. Certain food types (e.g., dairy and chocolate) and swabs/sponges benefit from an alternative enrichment strategy. See Appendix A, Alternative enrichment methods on page 25 for alternative sample preparation and enrichment recommendations for dairy, chocolate, and acidic/alkaline samples. 1. Food Samples a. Prepare a 1 in 10 dilution of the food sample in prewarmed Buffered Peptone Water (BPW) in a sterile bag with an internal strainer (e.g., Seward Product Code BA6041/STR). For example, add 25 g of food sample to 225 ml of BPW or add 325 g of food sample to 2925 ml of BPW. Note: Media should be prewarmed to 37±1 C prior to use. A standard Stomacher sterile bag or equivalent can be used with liquid samples. b. Homogenize the sample by stomaching the bag for about 1 minute at normal speed. Alternatively, hand mix for about 1 minute, massaging the sample between the fingers to disperse any large clumps of material (Narang and Cray, 2006). Environmental contact swabs a. Wipe an area with a prewetted swab. Note: Rehydrate swabs with about 0.5 ml of Dey-Engley Neutralizing Broth, or equivalent. b. Transfer swab to a sterile 15-mL tube containing 10 ml of Buffered Peptone Water prewarmed to 37±1 C and twirl in the broth for about 60 seconds. Environmental contact sponge samples a. Wipe an area with a prewetted sponge. Note: Rehydrate sponges with about 10 ml of Dey-Engley Neutralizing Broth, or equivalent. b. Transfer the sponge to a sterile Stomacher bag containing 100 ml of Buffered Peptone Water prewarmed to 37±1 C. c. Homogenize by stomaching the bag for about 1 minute at normal speed. Alternatively, hand mix for about 1 minute, massaging the sample between the fingers. Not included in the AOAC validation studies. 12 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

13 Chapter 2 Methods Sample pooling 2 2. Incubate at 37±1 C for 20±2 hours. Note: We recommend processing these sub-samples for pooling immediately. If storage of sub-samples is required, store at 5±3 C for up to 32 hours. Rewarm samples to 37 C prior to analysis on the Pathatrix Auto Instrument. Store the remaining enriched sample at 5±3 C for up to 32 hours, for potential reanalysis. Sample pooling 1. Remove the Sample and Elution Vessels from the Pathatrix consumable kit packaging and place into the Sample Vessel Holder (included with the Pathatrix Auto Instrument; Cat. no. ATUBEHOLD). 2. Partially remove the lids from both vessels, making a large enough opening to allow the addition of sample and wash buffer to the vessels. 3. Prepare a single pooled sample by transferring 5-mL aliquots from ten (10) individually enriched samples to the Sample Vessel. Note: If the samples are highly particulate and/or contain a high fat content, the use of foam filters (Cat. no. PFF) with pooling syringes and straws (Cat. no. POOL1010MLN) is recommended. 10X Individual Samples Individual Enrichment of Samples Postenriched Samples Pooling of Samples (10X 5-mL Samples) 50-mL Pooled Sample (1X Pathatrix Test) Agar plate If pooled sample is negative, then all 10 individual samples are negative. PCR: 7500 Fast Instrument & MicroSEQ pathogen detection kits If pooled sample is positive, then retest all 10 individual samples. Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 13

14 2 Chapter 2 Methods Samples and consumable loading 4. Store the individual enriched samples at 5±3 C for potential reanalysis until the test result is confirmed. Note: Do not store enriched samples for more than 32 hours. Samples and consumable loading 1. Add about 35 ml of 1X PBS (Cat. no. AM9624, diluted with sterile water to 1X) to the fill line of the Elution Vessel. 2. Replace the lids on the Sample and Elution Vessels, making sure that the vessels are completely sealed. 3. Add 50 µl of the Pathatrix bead suspension (Cat. no. APS500P) into the spout on the lid of the Sample Vessel. IMPORTANT! Fully resuspend the Pathatrix beads by agitating the bead vial (e.g., vortexing or inversion of sealed bead vial) before adding to the Sample Vessel. Sample Vessel Spout Sample Vessel Elution Vessel Sterile 1X PBS (35 ml) Sample Vessel Holder (ATUBEHOLD) 14 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

15 Chapter 2 Methods Samples and consumable loading 2 4. Remove the capture phase kit from the bag and orientate with the valve plunger pointing left. Connect the valve firmly to the lids of the Sample and Elution Vessels. Capture Phase Valve Plunger 5. Holding both vessels, lift the assembled vessels and attached kit out of the Sample Vessel Holder. 6. Place the vessels into the Cartridge, pushing the vessels firmly in place from the bottom upwards. 7. Firmly push the remainder of the kit into the Cartridge, ensuring that the valve, phase, and syringe are all held securely in the molded recess of the Cartridge. Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 15

16 2 Chapter 2 Methods Sample unloading 8. Push the magnet slider across into the locking position and press the magnet release button on the side of the Cartridge to ensure the kit is positioned correctly and the magnets can freely disengage from the phase. Syringe Magnet Release Button Phase Sliding Magnet Holder Valve Plunger Sample Vessel Elution Vessel Cartridge Holder (ACARTHOLD) 9. Reset the magnets into the locking position. 10. Insert the Cartridge into the Pathatrix Auto Instrument until it clicks into the locking position. 11. Press the numbered button above the appropriate Cartridge to start the run. The associated LED will turn green to indicate the run has started. Sample unloading 1. At the end of the run, the LED flashes red and green alternately. 2. Press the button above the appropriate Cartridge to initialize the draining step (approximately 1 minute). 3. When the draining step is complete and the LED is illuminated red, remove the Cartridge by pulling it out, away from the instrument. 16 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

17 Chapter 2 Methods DNA sample preparation for real-time PCR 2 4. Remove the syringe from the Cartridge and carefully pull out the rest of the kit, starting at the top and working downwards. When removing the Sample and Elution Vessels from the Cartridge, hold firmly by the vessels themselves to prevent spillage. 5. Place both vessels with the capture phase attached in the Sample Vessel Holder. 6. Remove the lid from the Elution Vessel to separate it from the rest of the consumable. 7. Leaving the Elution Vessel in the Sample Vessel Holder, lift away and discard the rest of the consumable, including the Sample Vessel. Note: The Elution Vessel contains approximately 200 µl of PBS, magnetic beads, and the captured bacteria. The Pathatrix bead suspension is ready for immediate analysis by real-time PCR. DNA sample preparation for real-time PCR Note: DNA sample preparation requires one heating block for incubating 1.5 ml tubes. The heat block should be set at 97±2 C. Alternatively, if using PCR plates, the incubation can be performed in a thermal cycler, if available. 1. Prefill a sufficient number of 1.5 ml centrifuge tubes or wells of a 96-well PCR plate each with 10 µl of 1X Lysis Buffer, FS (Cat. no ) to correspond to all samples being tested, including any needed negative controls. 2. Allow the Elution Vessel to sit in the Sample Vessel Holder for approximately 1 minute to allow capture of the Pathatrix beads. Note: If more than one Cartridge is run, Elution Vessels may be transferred to an Elution Vessel Holder (included with the Pathatrix Auto Instrument; Cat. no. ATUBERACK). Both the Sample Vessel Holder (holds 1 Elution Vessel) and the Elution Vessel Holder (holds up to 5 Elution Vessels) contain magnets to capture beads on one side of the bottom of the Elution Vessels. 3. While the Elution Vessel is still in the vessel holder, carefully remove and discard the PBS from the Elution Vessel, taking care not to disturb the captured Pathatrix beads. Note: (Optional) Some sample types will carry over large amounts of debris into the elution tube. If, when removing the PBS from the elution tube, you notice that the solution is not clear, we recommend that you perform a manual PBS wash on the beads to prevent lysis and PCR inhibition. Perform this secondary wash step as follows: a. Remove the Elution Vessel from the holder and add 120 µl clean 1X PBS (Cat. no. AM9624 diluted with water to 1X). Pipet up and down to wash and fully resuspend the beads. b. Place the Elution Vessel back in the vessel holder and allow it to sit for approximately 1 minute to allow capture of the Pathatrix beads. c. Carefully remove and discard the PBS wash from the Elution Vessel, taking care not to disturb the captured Pathatrix beads. Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 17

18 2 Chapter 2 Methods DNA sample preparation for real-time PCR 4. Remove the Elution Vessel from the vessel holder and completely resuspend the Pathatrix beads in 120 µl of Molecular Biology Grade water by pipetting up and down. 5. Transfer 90 µl of the resuspended Pathatrix bead pellet to a 1.5-mL microcentrifuge tube which already contains 10 µl of 1X Lysis Buffer, FS. Note: If preparing many samples, the samples can be transferred to the appropriate wells of a 96-well PCR plate. After loading the 96-well plate with all samples, seal the plate with non-optical film. Use a heat block compatible with 96-well plates or a thermal cycler for the 97±2 C incubation. 6. The remaining 30 µl of the bead resuspension may be retained for further testing by direct plating on selective agar plates, if desired. a. If the sample is not going to be plated immediately, transfer the full 30 µl into a clean 1.5 ml microcentrifuge tube, and store at 5±2 C for no more than 24 hours. Be sure to store the tube(s) away from magnets. b. Streak about 10 µl onto each selective plate using a clean inoculating loop. If the sample was stored at 5±2 C, be sure that the sample has been resuspended before removing the 10 µl aliquots. c. Incubate plates at 37±1 C for hours. Refer to Interpretation of positive results by plating and biochemical assays on page 22 of this manual and the plates' manufacturers guides for further instructions and how to interpret the results. 7. Incubate sample(s) at 97±2 C for 10 minutes in a heating block (or thermal cycler). 8. Remove the sample(s) from the heating block (or thermal cycler) and allow the sample(s) to cool to room temperature for about 1 minute. 9. Proceed in one of the following ways: Proceed directly to Real-time PCR detection on page 19, if storage of the samples before PCR is not required. or Process further for long-term sample storage as follows: a. Place the DNA samples in a magnetic particle concentrator (if using microcentrifuge tubes; Cat. no D) or magnetic plate (if using a 96-well plate; Cat. no. MAGNETICPLATE or AM10027). b. Leave the sample in the concentrator for 2±1 minute. c. Remove up to 60 µl of the sample from the top of the tube and add it to a clean microcentrifuge tube or well of a 96-well plate. Avoid the magnetic particle pellet and any lysis debris while removing the sample. d. Store the DNA sample below 18 C for up to 1 year. IMPORTANT! Do not freeze the DNA samples with Pathatrix beads. 18 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

19 Chapter 2 Methods Real-time PCR setup 2 Real-time PCR setup Note: The RapidFinder Express Software is designed for use on the Applied Biosystems 7500 Fast Real-Time PCR System and must be set up before aliquoting samples. The RapidFinder Express Software provides online, step-by-step instructions to set up the assays followed by automated data analysis. For details, refer to the RapidFinder Express Software Online Help. 1. Create a run file document using the RapidFinder Express Software. The RapidFinder Express Software outlines sample placement in the 8-strip PCR reaction tubes. IMPORTANT! Within RapidFinder Express Software, refer to the Create or Edit Run File and Pipette Samples instructions in the RapidFinder Express Software Online Help. 2. From the MicroSEQ Salmonella spp. Detection Kit (Cat. no ), open the storage pouch containing the MicroSEQ assay beads by cutting at the notch in the upper corner of the storage pouch above the zip-lock strip. Note: Each real-time PCR sample tube contains a lyophilized assay bead that includes all the reagents needed for PCR, except for the unknown or control sample. IMPORTANT! Do not remove the desiccant from the storage pouch. 3. Remove the appropriate number of individual real-time PCR sample tubes or 8-tube strips, one tube for each reaction that you plan to run. Note: The 8-strip tubes can be cut apart using scissors, if fewer than 8 reactions are needed. 4. Place tubes on ice or in a cold block. 5. Seal the storage pouch using the zip-lock strip, then store at 5±3 C. Real-time PCR detection 1. Prepare the DNA samples from step 9 on page 18, as indicated in the following table, and dispense to the PCR sample tubes. Note: Dispense all unknown samples first, followed by negative control(s), and then positive control(s). You must follow the layout determined by the RapidFinder Express Software. Unknown samples and positive control samples are provided by the investigator. The MicroSEQ kits include a negative control (i.e., Pathogen Detection Negative Control). Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 19

20 2 Chapter 2 Methods Real-time PCR detection Frozen DNA samples (beads removed and DNA frozen as described in step 9 on page 18) DNA samples that have not been frozen (that is, with beads still present) 1. Just prior to use, completely thaw the DNA sample on ice. 2. Prior to opening, centrifuge briefly remove condensation from the tubes or plates and avoid cross contamination. 3. Transfer 30 µl of the thawed DNA sample to a realtime PCR sample tube from the MicroSEQ Salmonella spp. Detection Kit. 1. Place the DNA samples in a magnetic particle concentrator (if using microcentrifuge tubes; Cat. no D) or magnetic plate (if using a 96-well plate; Cat. no. MAGNETICPLATE or AM10027). 2. Leave the sample in the concentrator for 2±1 minute. 3. Carefully remove 30 µl from the top of the sample with a pipette and add it to a real-time PCR sample tube from the MicroSEQ Salmonella spp. Detection Kit. Avoid the magnetic particle pellet and any lysis debris at the bottom of the tube. 4. (Optional) If desired, some of the remaining DNA can be stored for potential reanalysis. Transfer 30 µl of the remaining sample to a clean microcentrifuge tube, avoiding the magnetic particle pellet and any lysis debris at the bottom of the tube. Store the DNA sample below 18 C. 2. Mix samples or controls with the lyophilized MicroSEQ assay beads by gently aspirating and dispensing a few times. (Alternatively, vortex the assay tubes after capping the tubes in step 4 on page 21.) Note: The MicroSEQ assay beads in the real-time PCR sample tubes dissolve in 1 5 seconds and contain all the components necessary for each reaction, except for the unknown or control sample. IMPORTANT! Follow these tips when setting up your PCR: Use a new pipette tip for each sample. Resuspend by gently pipetting up and down with the pipette tip at the bottom of the tube to minimize aerosol formation and cross-contamination or, if a vortexer is available, vortex the capped tubes until the pellet dissolves. Cap the tubes, sealing each tube with the transparent, optical strip-caps provided in the kit. Do not use colored caps or tubes for kit reactions. Colored caps or tubes may affect dye-signal readings during real-time PCR. For more information about Good PCR Practices, see Appendix C on page If the 8-strip tube was cut with scissors because <8 samples are being tested, then combine sample tubes with additional empty tubes to create a full set of 8 tubes prior to inserting caps and placing in the PCR instrument. Note: A complete set of capped, 8-tube strips evenly distributes the clamping load that is applied to the sample tube strips during processing, thereby minimizing the risk of collapsing any tubes. 20 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

21 Chapter 2 Methods Real-time PCR detection 2 4. Cap the tubes, sealing each tube with the transparent optical strip caps provided in the kit. Cap the tubes with the strip caps using the MicroAmp 96-Well Base (Cat. no. N ) and the MicroAmp Cap Installing Tool (Handle, Cat. no ) 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. 5. Mark or label one end of the strip cap (but not directly over any one cap) so that the orientation is conserved when transferring the tubes to the instrument tray. 6. Make sure reactions are thoroughly mixed and at the bottom of the tubes. In order to ensure that the entire sample is at the bottom of the tube, we highly recommend that you spin down the tube contents at 2000 g for 20 seconds using a centrifuge with a plate adapter or a benchtop microcentrifuge with an 8-strip PCR tube adapter. 7. To load the instrument, transfer the tubes to the 7500 Fast Instrument in the same configuration as the run layout. Use the 7500 Fast Precision Plate Holder for MicroAmp Tube Strips (Cat. no ; see the following figure). 8. Close the tray to the 7500 Fast Instrument (see the following figure). 9. When finished with the pipetting procedure, the software will prompt you for the next step (for example, Start Instrument Run), or click Close to return to the Main page. Press the tray door to open it. Transfer the tubes to the precision plate holder (PPH) in the tray. Close the tray door. Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 21

22 2 Chapter 2 Methods Retesting of individual samples after a positive pooled sample 10. Refer to the RapidFinder Express Software Online Help for information about: Selecting the run file. Performing the run. Viewing your results. Analyzing your data. 11. Proceed as indicated in the following table, according to the RapidFinder Express result. Result icon Result Positive result Negative result Result warning Action Pooled samples: proceed to Retesting of individual samples after a positive pooled sample on page 22. Individual samples: proceed to Interpretation of positive results by plating and biochemical assays on page 22. Proceed to Test result interpretation and classification on page 23. Proceed to Result warnings and recommendations for retesting on page 24. RapidFinder Express displays results pictorially. Retesting of individual samples after a positive pooled sample Some applications require retesting of individual samples to determine which sample was the cause of the positive pooled sample. Retest individual samples as follows. 1. Rewarm individual samples from step 4 on page 14 to 37±1 C, then remove 10 ml and transfer to a Sample Vessel. 2. Proceed immediately to Samples and consumable loading on page 14 and follow the procedure through Interpretation of positive results by plating and biochemical assays on page 22. Interpretation of positive results by plating and biochemical assays Samples identified as PCR-positive may be confirmed by plating retained unlysed Pathatrix beads from step 6 on page 18 (DNA sample preparation for real-time PCR) onto selective agar plates, followed by biochemical and serological methods (ISO 6579:2002 or FDA BAM Chapter 5). Note: If individual samples are used for confirmation, confirmation using pooled samples is optional. If PCR is not used to identify Salmonella-positive samples, then the beads from pooled samples or individual samples can be plated directly onto selective agar plates [Pub. no. MAN (10-pooled sample) or MAN (individual samples)] and confirmed by biochemical and serological methods (ISO 6579:2002 or FDA BAM Chapter 5). 22 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

23 Chapter 2 Methods Test result interpretation and classification 2 1. Streak 10 µl (one loopful) of the unlysed Pathatrix bead suspension from step 6 on page 18 onto a well-dried XLD agar plate, and streak an additional 10 µl onto a second selective agar plate of choice. IMPORTANT! We recommend streaking, instead of spreading, to generate isolated colonies. Note: The second selective agar plate can be any other solid selective medium complementary to XLD as long as it is appropriate for the isolation of lactosepositive Salmonella, Salmonella typhi, and Salmonella paratyphi strains. Refer to the media section of Required materials not included in the kits on page 9 for examples. Incubate the second selective agar plate according to manufacturer s instructions. 2. Allow the plates to dry for 10 minutes, then invert and incubate at 37±1 C for hours. Note: A presumptive positive is identified as any typical or atypical growth on the selected agar plates that would indicate Salmonella spp. colonies. Any presumptive positive isolate should be subsequently confirmed by sub-culture, followed by the appropriate biochemical and serological tests detailed in ISO 6579:2002. Test result interpretation and classification Once confirmed, the results are reported as: Salmonella spp. Detected in g (sample matrices) Salmonella spp. Not detected in g (sample matrices) Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 23

24 2 Chapter 2 Methods Result warnings and recommendations for retesting Result warnings and recommendations for retesting Result Warnings are shown when the internal positive control (IPC) in a reaction fails to amplify, and the target sequence also fails to amplify. Result Warnings are usually the result of PCR inhibition due to carry over of inhibitors from the sample preparation steps of the workflow. If a Result Warning is obtained on a sample, re-test that sample at a 1:6 dilution using the following procedure. Note: For more information about how to view and interpret amplifications plots (optional), see the Appendix, "Investigating Warning Results or Failed Runs in the SDS Software". 1. Prepare for real-time PCR as described in Real-time PCR setup on page Retrieve the retained DNA sample that was stored as described in step 1 in Realtime PCR detection. Fully thaw the DNA sample on ice, if necessary. 3. Dispense 25 µl of microbiology grade water to a real-time PCR sample tube from the MicroSEQ Salmonella spp. Detection Kit, and immediately add 5 µl of the thawed DNA sample. 4. Dispense negative control(s) and positive control(s) following the layout determined by the RapidFinder Express Software. Note: Dilution of the controls is not required. 5. Mix samples or controls with the lyophilized MicroSEQ assay beads as described in step 2 of Real-time PCR detection, and continue the Real-time PCR detection procedure. 24 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

25 A Alternative enrichment methods See AOAC Performance Tested MethodsSM Certification on page 35 for details about matrices used in the AOAC validation study. Prepare potentially acidic/alkaline samples for enrichment Samples which potentially deviate from neutral ph (for example, milk powders, fermented milk samples, etc.) should be prepared as follows: 1. Prepare the sample according to the sample enrichment protocol. 2. Incubate for 60±5 minutes at room temperature. 3. Homogenize the sample and determine the ph. 4. If necessary, adjust the ph to 6.8±0.2, and mix well before determining the final ph. Dairy samples Dairy samples include milk-derived products such as: Fermented milk products (cheeses, yogurt) Dry milk products (infant milk powder, non-fat powdered milk) Liquid milk products (pasteurized milk) Raw milk products (unpasteurized cheeses, liquid raw milk) Dairy and milk powder samples can benefit from an alternative enrichment strategy. For dairy samples, use prewarmed Buffered Peptone Water (BPW) supplemented with Brilliant Green (0.002%) as the enrichment media. Additionally, see the section Prepare potentially acidic/alkaline samples for enrichment. Incubate dairy samples at 37 C for 20±2 hours. Prewarm the BPW supplemented with Brilliant Green to 37±1 C prior to use. Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 25

26 A Appendix A Alternative enrichment methods Chocolate and cocoa-based samples Chocolate and cocoa-based samples All chocolate or cocoa-based samples can benefit from an alternative enrichment. Use prewarmed sterile UHT skim milk (or non-fat dry milk reconstituted in Molecular Biology Grade water) supplemented with Brilliant Green (0.002%), instead of Buffered Peptone Water, as the enrichment media. Incubate chocolate and cocoa-based samples, as described in the protocol, at 37 C for 20±2 hours. Prewarm the UHT skim milk supplemented with Brilliant Green to 37±1 C prior to use. 26 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

27 B Troubleshooting General troubleshooting Observation Possible cause Recommended action Enriched culture is curdled. PBS removed from the captured Pathatrix beads is not clear. In positive-control wells, no IPC signal is detected, but target-specific signal is detected. In positive-control wells, no target-specific signal is detected. In negative control wells, target-specific signal is detected. Samples may be slightly acidic or alkaline (for example, milk powders, fermented milk samples, etc.). Debris from the food sample has been carried over into the elution tube. A high copy number of target DNA exists in samples, resulting in preferential amplification of the target-specific DNA. Positive control was omitted (pipetting error). Carryover contamination occurred. 1. Mix the food sample with appropriate enrichment media. 2. Incubate for 60±5 minutes at room temperature (23±5 C). 3. Homogenize the sample and determine the ph. 4. Adjust the ph to 6.8±0.2, and mix well before determining the final ph. Perform a manual PBS wash on the Pathatrix beads as described in DNA sample preparation for real-time PCR on page 17. No action is required. The result is considered positive. Repeat the assay. Make sure to pipet positive control into all positive-control wells. 1. Repeat the assay using fresh aliquots of all reagents and clean pipetting equipment. 2. If the negative control continues to show contamination, repeat the assay using a new kit. 3. If the negative control continues to show contamination, contact Life Technologies Technical Support. Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 27

28 B Appendix B Troubleshooting General troubleshooting Observation Possible cause Recommended action In negative-control wells, no IPC is detected, but targetspecific signal is detected. In unknown sample wells, no IPC is detected, but targetspecific signal (C t <35) is detected. Multicomponent Plot signals for FAM, VIC, and ROX dyes increase/decreases during cycles 1 15, but the overall curve and result is not affected, when using View in SDS mode to analyze results. Replicate results for this sample are inconsistent. Carryover contamination caused target signal in negative-control wells. Additionally, no IPC signal in negative-control wells can be caused by: 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 A high copy number of target DNA exists in samples, resulting in preferential amplification of the target-specific DNA. Incomplete mixing and dissolution of the lyophilized bead with sample or control. All replicate wells for a sample do not have the same result. To correct carryover contamination, repeat the assay using fresh aliquots of all reagents and clean pipetting equipment. To determine whether IPC amplification is a problem, examine unknown wells for an IPC signal. If an IPC signal is present, IPC amplification is not a problem. No action is required. The result is considered positive. After addition of 30 µl of sample or Pathogen Negative Control to the beads and capping the tubes: 1. Vortex strips at high speed for about 10 seconds and centrifuge the strips at g for about 10 seconds. 2. Vortex the strips again at high speed for about 10 seconds, and centrifuge the strips at g for about 1 minute. Ensure that all liquid is at the bottom of the tubes and the beads are fully dissolved before proceeding with the PCR. 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 you ran only two replicates and results are not consistent, repeat the assay using fresh samples and reagents. 28 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

29 Appendix B Troubleshooting Investigating warning results or failed runs in the SDS Software B Observation Possible cause Recommended action Amplicon contamination. Contamination was introduced into the PCR clean area from postamplification reaction tubes that were either opened in the clean area or brought into the PCR clean area from contaminated gloves or solutions. Contamination was introduced into the real-time PCR instrument from crushed and broken PCR tubes. To confirm amplicon contamination, perform the following experiment: Prepare negative control samples using at least one 8-tube strip of MicroSEQ Assay Beads. 1. Divide the number of assay beads into 2 sets. a. To the first set of assay beads, add 30 µl of nuclease-free water. b. To the second set of assay beads, add 29 µl of nuclease-free water plus 1 µl of 1 U/µL Uracil DNA Glycosylase (Cat. no ). 2. Run samples on the 7500 Fast Real-Time PCR Instrument using SDS software and select the Fast 7500 Run Mode. 3. Under the instrument tab: Select Add Step to stage 1 of the PCR cycle that consists of 10 minutes at 50 C. Extend the 95 C step from 20 seconds to 10 minutes. Amplicon contamination is indicated by target-specific signal in the UNG samples and no target-specific signal in +UNG samples. 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 ) and/or contact a service representative to clean the instrument. Investigating warning results or failed runs in the SDS Software IMPORTANT! If you modify a RapidFinder Express Software run file in the SDS Software, you cannot open the run file again in the RapidFinder Express Software. To avoid altering a RapidFinder Express Software run file, save the run file under a new name in the SDS Software before performing any actions, as described below. 1. Open the run file in the SDS Software by one of the following methods: From the View Results page in the RapidFinder Express Software, select the run file, then click View in SDS. Open the run file in the SDS Software. 2. Select File Save As, then save the run file under a new name. 3. Select the Results tab. Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 29

30 B Appendix B Troubleshooting Investigating warning results or failed runs in the SDS Software 4. Select the Amplification Plot tab. 5. Select all locations by clicking the top left corner of the layout. 6. Examine the Amplification Plot in Data mode of Delta Rn vs. Cycle (displayed by default). To examine the signal for only tubes of interest, Ctrl + Click the locations below the plot. To examine the signal for only the IPC or a specific target, select the signal of interest from the Detector list at the top right of the plot. Refer to the following section, Interpretation of amplification plot for samples with a Result Warning. 7. When you finish viewing the run file, exit the SDS software: If you accessed the run file from the RapidFinder Express software, in the SDS Software, select File Return to RapidFinder Express Software. If you opened the run file directly in the SDS software, in the SDS Software, select File Exit. 30 Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide

31 Appendix B Troubleshooting Interpretation of amplification plot for samples with a Result Warning B Interpretation of amplification plot for samples with a Result Warning For the IPC and the target (Salmonella spp.) detector, observe if the curve displayed in the Amplification Plot crosses the highlighted horizontal line, sometimes referred to as the threshold line. IMPORTANT! The RapidFinder Express software will automatically select the appropriate threshold values for each detector. Unless advised by a Life Technologies representative, do not change these values. Additionally, the highlighted horizontal line, as described above, will appear only when one of the detectors is selected. If All detectors are selected, the horizontal line will not be in the correct location for proper visual identification of the sample(s). Observation Interpretation Recommended action IPC curve does not cross the threshold line IPC and Target curves do not cross the threshold line IPC curve does cross the threshold line Target curve does not cross the threshold line IPC in this sample was inhibited PCR inhibition in the sample No PCR inhibition Sample is negative for target detection Dilute sample lysate 1:6 and re-run; see Result warnings and recommendations for retesting on page 24 for details. None needed; result is negative Pathatrix 10-Pooling Salmonella spp. Kit Linked to MicroSEQ Salmonella spp. Detection Kit User Guide 31