Pathatrix® Listeria spp. Kit (Individual Samples) Linked to PCR and
Contents
1. Listeria spp Kit Linked to the Idaho Technology R A P I D LT PCR System Detection direct plating optional Review the amplification curves and melt peaks as described in the R A P D 9 LT PCR software Screen Shot Protocol If any Amplification Curve exhibits an increase in fluorescence and or displays a potential Melt Peak in the absence of a positive PCR software determination a repeat PCR analysis should be carried out by repeating the R A P D 9 LT PCR steps in this section see page 26 If a positive PCR result is obtained an aliquot of the Pathatrix beads should be plated out see the following section Detection direct plating optional Detection direct plating optional Note We recommend streaking the sample out to generate individual colonies as opposed to spread plating 1 Pipette the remainder of the Pathatrix9 bead suspension onto the edge of well dried PALCAM and Listeria Oxford or equivalent agar plates e g Chromogenic Listeria agar ALOA 2 Using a sterile 10 uL inoculation loop streak from this pool to generate isolated colonies 3 Allow the plates to dry for approximately 10 minutes then invert and incubate at 37 1 C for 18 48 hours as appropriate 24 48 hours was used in the AOAC RI validation study A presumptive positive result is defined as the isolation of typical suspicious or atypical Listeria spp colonies on the agar plates used A presumptive positive isolate2. conditions The genus Listeria is composed of six species L monocytogenes L innocua L welshimeri L seeligeri L ivanovii L grayi and two recently discovered new species given the provisional names L marthii and L rocourti 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 The Pathatrix Listeria spp Kit is for professional use only and is intended for use by qualified users interested in determining the presence absence of Listeria spp in food samples Users may include but are not limited to food producers food processors food manufacturers retailers and microbiology testing laboratories The sample preparation procedure allows you to detect as few as 1 10 cfu from 25 grams of food sample The limitation of the Pathatrix Listeria spp Kit is in the ability of the target to reproduce in the enrichment medium be captured by the magnet and subsequently be detected by BAX PCR R A P 1 D9 LT PCR or the VIDAS immunoassay testing systems or be isolated on selective agar plates CAUTION The Pathatrix
3. should be subsequently confirmed by the use of subculture and appropriate biochemical and serological tests as required Test result interpretation and classification The Pathatrix Listeria spp Kit is designed as a sample preparation method for presence absence detection of Listeria spp in food matrices Using the Pathatrix Listeria spp Kit linked to the R A P D LT PCR system presumptive results can be obtained prior to confirmation within 25 27 hours Once confirmed the results are reported as e Listeria spp Detected in 25 g sample matrices e Listeria spp Not detected in 25 g sample matrices Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 27 2 Chapter 2 Pathatrix Listeria spp Kit Linked to the Idaho Technology R A P 1 D LT PCR System Test result interpretation and classification 28 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Workflow Pathatrix Listeria spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System Sample enrichment page 30 v Sample preparation page 31 h Samples and consumable loading page 32 vy Sample unloading page 35 h Detection VIDAS immunoassay testing protocol page 36 vy Detection direct plating optional page 37 vy Test result interpretation and classification page 37 Procedural guidelines e Use aseptic technique and good laboratory practices at all times e A facemask shou
4. Oxoid product code SRO141E For optional secondary enrichment MOPS BLEB Oxoid product code CM1071 Selective agar PALCAM agar base Oxoid product code CM0877 PALCAM selective supplement Oxoid product code SRO150 Listeria selective agar Oxford formulation Oxoid product code CM0856 Listeria selective supplement Oxford Oxoid product code SRO140 formulation Modified Listeria selective supplement Oxoid product code SRO206 Oxford formulation Alternative chromogenic agar Brilliance Listeria agar base Oxoid product code CM1080 Brilliance Listeria selective supplement Oxoid product code SRO227 Reagents PBS 10X pH 7 4 Life Technologies Cat no AM9624 or AM9625 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 7 Product Information Materials not included in the kit 8 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Workflow Pathatrix Listeria spp Kit Linked to the DuPont BAX PCR System Sample enrichment page 10 v Sample preparation page 11 h Samples and consumable loading page 12 vy Sample unloading page 15 v Detection BAX PCR protocol page 16 h Detection direct plating optional page 17 vy Test result interpretation and classification page 17 Procedural guidelines Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA e Use aseptic technique and good laboratory practices at all times e A face
5. SRO166E or equivalent e g BLEB c Homogenize the sample by hand mixing for 10 15 seconds Massage the sample between the fingers to disperse any large clumps of material Narang and Cray 2006 Alternative For enrichment of environmental contact swabs sterile 15 mL tubes may be used as an alternative to sterile bags a Add 10 mL of pre warmed media to a sterile 15 mL tube b After swabbing an area twirl swab in the media in the 15 mL tube for approximately 1 minute Alternative Environmental contact sponges may be used as an alternative to swabs a Wipe of an area with a rehydrated pre wetted sponge and transfer the sponge to a sterile bag Note Sponges may be pre wetted with 10 mL of Dey Engley Neutralizing Broth D E broth or enrichment media b Add 100 mL of pre warmed 37 1 C appropriately supplemented Half Fraser broth Oxoid CM0895 with supplement SRO166E or equivalent e g BLEB c Homogenize the sample by hand mixing for approximately 1 minute Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Chapter 2 Pathatrix Listeria spp Kit Linked to the Idaho Technology R A P I D LT PCR System Sample preparation 2 Incubate ALL samples at 37 1 C for a minimum of 24 hours Note We recommend that these sub samples for analysis are processed immediately or if storage is required that the samples are refrigerated at 5 3 C Samples should be rewarmed to 37 1 C prior
6. kit has been evaluated on soft cheese cream and cooked chicken food matrices Given the wide variety of products and manufacturing procedures we recommend that you check that the composition of the matrices to be tested does not affect the reliability of the results A negative result does not guarantee the absence of target organism in the original sample and may be due to the inability of the organism to adequately reproduce to required levels in the enrichment medium with subsequent outgrowth on selective agar plates potentially due to but not limited to competitive microflora sub lethal injury or matrix inhibition The Pathatrix Auto Instrument is for indoor use only and for altitudes not exceeding 2000 m 6500 ft above sea level Temperature and humidity requirements Condition Acceptable range Temperature 5 to 40 C Humidity Maximum relative humidity 80 for temperatures up to 31 C decreasing to 50 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 39 C Ordering Information Related materials from Life Technologies Equipment consumables and reagents Item Cat no Related consumable kits with associated beads Pathatrix 5 Pooling Listeria spp Kit APL250P Pathatrix 10 Pooling Listeria spp Kit APL500P Pathatrix DUAL Salmonella spp Listeria spp Kit APD50 Equipment Pathatrix Auto Instrument PATHATRIXAUTO Cartridge Rack optiona
7. rack and cap with a flat lid provided in the consumable kit Leave in the rack for 1 minute to allow capture of the beads 8 Remove all the liquid from the Elution Vessel without removing the vessel from the rack taking care not to disturb the captured beads 9 Add 100 uL of PBS into the Elution Vessel and resuspend the Pathatrix beads 10 Appropriate aliquots of the Pathatrix bead suspension can then be immediately analyzed using the laboratory s chosen pathogen detection method Note The bead suspension may be retained for later testing if necessary The beads should be stored in sterile 1 5 mL microcentrifuge tubes AWAY from magnets e g away from Pathatrix vessel holders at 5 3 C for up to 24 hours Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 25 2 Chapter 2 Pathatrix Listeria spp Kit Linked to the Idaho Technology R A P 1 D LT PCR System Detection R A P1 D 9 LT PCR protocol Detection R A P I D LT PCR protocol IMPORTANT It is important and entirely the operator s responsibility to refer to the package insert instructions and instrument user manual for information regarding the correct set up and operation of the R A P 1 D LT PCR system 1 Add 25 uL of the resuspended Pathatrix beads into a Blue Capped Bead Tube and vortex on the Disruptor Genie vortexer equipped with the TurboMix attachment for 5 minutes on the highest setting 2 Once
8. the bead beating lysis step is complete immediately place the Bead Tubes into the DynaMag 2 Magnet Cat no 123 21D 3 Wait for at least 1 minute to allow the Pathatrix bead debris to be drawn out of suspension thereby producing the bead free supernatant Note If present target DNA will be in the bead free lysate 4 Pipette 10 uL of Reconstitution Buffer into the Unknown test vial 5 Pipette 10 uL of bead free supernatant into the PCR Unknown test reaction vial Mix well to fully resuspend the PCR reagent pellet Note Take care not to transfer any glass beads from the Blue Capped Bead Beating Tubes into the PCR reagent vial 6 Transfer 18 uL of the reagent sample suspension and add into the top of a glass PCR capillary tube Using the Capping Tool provided place the cap on the sample capillary tube and push down firmly 7 To proceed refer to the R A P 1 D 9 LT PCR operating instructions nw Standard sample 25 uL V Add 100 pL PBS to resuspend the Transfer 25 uL of bead suspension Bead beat for 5 min to release Capture beads for 2 min Pathatrix beads into Small Bead Tube with glass target DNA beads 5 40 uL Bead free supernatant Aliquot bead free supernatant Proceed to PCR platform e g 5 40 uL into PCR reaction RAPI D LT BAX iCycler or mixture LightCycler instrument 26 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Chapter 2 Pathatrix
9. to analysis on the Pathatrix Auto Instrument The remaining enriched sample should be stored until the results have been determined Sample preparation 1 Remove the Sample and Elution Vessels from the consumable kit packaging and place into the Sample Vessel Holder Partially remove the lids from both vessels making a large enough opening to allow the sample and wash buffer to be dispensed into the vessels Food samples Prepare a 50 mL sample in the Sample Vessel by transferring a 50 mL aliquot from the enriched sample Swab samples Prepare a 10 mL sample in the Sample Vessel by transferring a 10 mL aliquot from the enriched sample Sponge samples Prepare a 10 mL sample in the Sample Vessel by transferring a 10 mL aliquot from the enriched sample Note If the samples are highly particulate and or contain a high fat content a Seward plain sterile bag with internal strainer may be used Seward Product Code BA6041 STR or equivalent Store the individual enriched samples at 5 3 C for potential reanalysis until the test result is confirmed Note Do not store for more than 32 hours If refrigerated after enrichment samples should be rewarmed to 37 1 C prior to removal of aliquots for analysis Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 21 2 Chapter 2 Pathatrix Listeria spp Kit Linked to the Idaho Technology R A P 1 D LT PCR System Samples and consumable loading Samples and
10. 0 mL of pre warmed 37 1 C appropriately supplemented Half Fraser broth Oxoid CM0895 with supplement SRO166E or equivalent e g BLEB c Homogenize the sample by hand mixing for approximately 1 minute Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Chapter 3 Pathatrix Listeria spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System Sample preparation 2 Incubate ALL samples at 37 1 C for a minimum of 40 hours Note We recommend that these sub samples for analysis are processed immediately or if storage is required that the samples are refrigerated at 5 3 C Samples should be rewarmed to 37 1 C prior to analysis on the Pathatrix Auto Instrument The remaining enriched sample should be stored until the results have been determined Sample preparation 1 Remove the Sample and Elution Vessels from the consumable kit packaging and place into the Sample Vessel Holder Partially remove the lids from both vessels making a large enough opening to allow the sample and wash buffer to be dispensed into the vessels Food samples Prepare a 50 mL sample in the Sample Vessel by transferring a 50 mL aliquot from the enriched sample Swab samples Prepare a 10 mL sample in the Sample Vessel by transferring a 10 mL aliquot from the enriched sample Sponge samples Prepare a 10 mL sample in the Sample Vessel by transferring a 10 mL aliquot from the enriched sample Note If t
11. 2 is used for processing 10 mL swab samples and can be selected by depressing the scroll arrow keys located either side of the LCD display simultaneously for approximately 5 seconds then pressing the left arrow key once to select P2 The display will now indicate that P2 has been selected by showing PATHATRIX P2 SELECTED Note To return to the default P1 run setting for processing food samples again press both arrow keys for 5 seconds then press the RIGHT arrow key to select P1 The display will then return to the standard PATHATRIX screen as normal Insert the Cartridge into the Pathatrix9 Auto Instrument until it clicks into the locking position Press the numbered button above the appropriate Cartridge to start the run The associated LED will turn green to indicate the run has started P1 approximately 14 minutes P2 approximately 25 minutes Program P2 is used to process 10 mL environmental samples and is not AOAC validated If an AOAC validated protocol is desired environmental samples may be analyzed using P1 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Chapter 3 Pathatrix Listeria spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System Sample unloading 10 11 12 13 Sample unloading Atthe end of the run the LED will flash red and green alternately Press the button above the appropriate Cartridge to initialize the draining step approxi
12. CR System 1 Detection direct plating optional Detection direct plating optional Note We recommend streaking the sample out to generate individual colonies as opposed to spread plating 1 Pipette the remainder of the Pathatrix bead suspension onto the edge of well dried PALCAM and Listeria Oxford or equivalent agar plates e g Chromogenic Listeria agar ALOA 2 Using a sterile 10 uL inoculation loop streak from this pool to generate isolated colonies 3 Allow the plates to dry for approximately 10 minutes then invert and incubate at 37 1 C for 18 48 hours as appropriate 24 48 hours was used in the AOAC RI validation study A presumptive positive result is defined as the isolation of typical suspicious or atypical Listeria spp colonies on the agar plates used A presumptive positive isolate should be subsequently confirmed by the use of subculture and appropriate biochemical and serological tests as required Test result interpretation and classification The Pathatrix Listeria spp Kit is designed as a sample preparation method for presence absence detection of Listeria spp in food matrices Using the Pathatrix Listeria spp Kit linked to the BAX PCR system presumptive results can be obtained prior to confirmation within 28 30 hours Once confirmed the results are reported as e Listeria spp Detected in 25 g sample matrices e Listeria spp Not detected in 25 g sample matrices Pathatri
13. Cartridge hold firmly by the vessels themselves to prevent spillage Place both vessels in the Tube Rack also known as the Sample Vessel Holder or rack Remove the lid from the Elution Vessel and leaving the Elution Vessel in place in the rack lift away the rest of the consumable including the Sample Vessel and discard Place the Elution Vessel in the rack and cap with a flat lid provided in the consumable kit Leave in the rack for 1 minute to allow capture of the beads Remove all the liquid from the Elution Vessel without removing the vessel from the rack taking care not to disturb the captured beads Remove the Elution Vessel from the vessel holder add 100 uL of PBS into the Elution Vessel and resuspend the Pathatrix beads Appropriate aliquots of the Pathatrix bead suspension can then be immediately analyzed using the laboratory s chosen pathogen detection method Note The bead suspension may be retained for later testing if necessary The beads should be stored in sterile 1 5 mL microcentrifuge tubes AWAY from magnets e g away from Pathatrix vessel holders at 5 3 C for up to 24 hours Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 15 Chapter 1 Pathatrix Listeria spp Kit Linked to the DuPont BAX PCR System Detection BAX PCR protocol Detection BAX PCR protocol IMPORTANT It is important and entirely the operator s responsibility to refer to the
14. Listeria spp Kit Individual Samples Linked to PCR or ELISA Chapter 1 Pathatrix Listeria spp Kit Linked to the DuPont BAX PCR System 1 Samples and consumable loading 6 Place the vessels into the Cartridge pushing them in firmly from the bottom upwards 7 Firmly push the rest of the kit into the Cartridge starting with the valve followed by the phase and finally the syringe 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 will move away from the phase freely Syringe Phase Sliding Magnet Holder Magnet Release Button Valve Plunger Sample Vessel Elution Vessel Cartridge Holder ACARTHOLD 9 Reset the magnets into the locking position Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 13 14 Chapter 1 Pathatrix Listeria spp Kit Linked to the DuPont BAX PCR System Samples and consumable loading 10 Food samples Program 1 P1 is used for processing 50 mL food samples and Pl is the Default mode on the Pathatrix9 Auto Instrument Swab samples Program 2 P2 is used for processing 10 mL swab samples and can be selected by depressing the scroll arrow keys located either side of the LCD display simultaneously for approximately 5 seconds then pressing the left arrow key once to select P2 The display will now indicate that P2 h
15. Product Code BA6041 STR or equivalent 4 Store the individual enriched samples at 5 3 C for potential reanalysis until the test result is confirmed Note Do not store for more than 32 hours If refrigerated after enrichment samples should be rewarmed to 37 1 C prior to removal of aliquots for analysis Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 11 1 Chapter 1 Pathatrix Listeria spp Kit Linked to the DuPont BAX PCR System Samples and consumable loading Samples and consumable loading 1 Add PBS Cat no AM9624 diluted to 1X to the fill line of the Elution Vessel about 35 mL of 1X PBS 2 Place the lids on to the Sample and Elution Vessels making sure that the vessels are sealed all the way around 3 Ensure the beads are fully resuspended by agitating the bead vial e g vortexing bead vial or inversion of sealed vial and add 50 uL of the bead suspension beads from Cat no APL50 into the spout on the lid of the Sample Vessel Sample Vessel Spout Elution Vessel Sample Vessel Sterile 1X PBS 35 mL Sample Vessel Holder ATUBEHOLD 4 Remove the plastic 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 wwe P ww Capture Phase Valve Plunger 5 Holding both vessels lift the assembled vessels and attached kit out of the Sample Vessel Holder 12 Pathatrix
16. Ss 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 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 41 Appendix D Safety Biological hazard safety 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 lim
17. T PCR protocol and VIDAS protocol DynaMag 2 Magnet Life Technologies Cat no 123 21D Forceps scissors spatula knife and or MLS scalpel Consumables Optional for environmental samples Swabs or sponges MLS 6 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Product Information Materials not included in the kit Equipment consumables and reagents Item Source Sterile bags for enrichment Whirl Pak or Nasco B01196WA Seward product code Stomacher bag or equivalent BA6041 or equivalent Optional for high particulate or high fat content samples Sterile filter bags for enrichment Whirl Nasco B01348WA Seward product code Pak or Stomacher bag or equivalent BA6041 STR or equivalent Microcentrifuge tubes PCR clean 1 5 mL MLS Sterile 10 uL disposable loops MLS Media See Sample Enrichment sections in the protocols page 10 20 or 30 and Appendix A on page 38 for recommendations about enrichment media choice The media is supplied by several manufacturers e g Oxoid product codes shown Difco and Merck in a dehydrated form and should be prepared according to the manufacturer s instructions Half Fraser broth Oxoid product code CM0895 Half Fraser broth supplement Oxoid product code SRO166E Alternative for environmental samples Buffered Listeria enrichment broth BLEB Oxoid product code CM0897 BLEB supplement
18. USER GUIDE applied biosystems oy Life technologies Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA For use with the Pathatrix Auto Instrument Catalog Number APL50 Publication Number MAN0007136 Revision 4 September 2012 o For testing of Food and Environmental samples only technologies 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 Environmental testing quality control quality assurance testing food and agricultural testing 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 re
19. ad A end ont Ach ene ant ta d 6 Materials not included in the kit 0 00 0 ccc ce cece eee ene ee eee eens 6 CHAPTER 1 Pathatrix Listeria spp Kit Linked to the DuPont BAX PCR System 0 cece eect o 9 WOPKTOW sci sostenida ii tits Petr ideo Risen tae 9 Procedural guidelines tito ata nunana naaran rrearen aeaaeae 9 Sample enrichmMent 0 2 seem ee gg e e FEE 10 Sample preparation Poe ey ed tl Phd ada de adel 11 Samples and consumable loading 00ceee eee cette ete teen eeeee 12 Sample unloading 242 2vo esesy booed ee dere ged a a bee eee Da ee eee Pee 15 Detection BAX PCR protocol 2 0 2 cn ncn n nen rrer n een eenes 16 Detection direct plating optional 0 ence nnn ene en ee nes 17 Test result interpretation and classification uues c cece eee eee eee eee eee ees 17 CHAPTER 2 Pathatrix Listeria spp Kit Linked to the Idaho Technology R A P I D LT PCR System 00000 cues 19 Work OW si soci races nes A Et sie be bw ae wed Seale eee 19 Procedural guidelines 0000 A A beer Meee Pade o eens 19 Sample enrichment cc 20 Sample preparation coa at A ens 21 Samples and consumable loading ooococccccccccr eect tet tee e teenies 22 Sample unloading ses ens elles cee ae es Be hake sae wee ao he eee eae eae 25 Detection R A PI D LT PCR protocol 2 0 0 0 ccc cnc rr 26 Detection direct plating optional 0 0 need nen een ee nes 27 Test result interpretat
20. apter 3 Pathatrix Listeria spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System Detection VIDAS immunoassay testing protocol Detection VIDAS immunoassay testing protocol IMPORTANT Itisimportant and entirely the operator s responsibility to refer to the package insert instructions and instrument user manual for information regarding the correct set up and operation of the VIDAS system 1 Ensure that the tubes are securely capped and then heat in a water bath at 95 C for 15 minutes to release target antigens 2 After heating immediately place the tube into the DynaMag 2 Magnet Cat no 123 21D for 1 minute to allow the Pathatrix beads to be pulled out of the lysate 3 Once the Pathatrix beads have aggregated against the magnet open the tube and remove 500 uL of the bead free supernatant and inoculate the VIDAS strip Note Target DNA if present will be in the bead free supernatant 4 To proceed with the test refer to the VIDAS operating instructions Standard sample 600 pL Add 700 pL of pre warmed BPW Transfer 600 uL of bead Vortex the tube for 10 sec and then Vortex the tube for 10 sec and then 37 C to resuspend the Pathatrix suspension into a standard 2 0 mL incubate the bead suspension for place in a water bath at 95 C and beads screw cap microfuge tube 2 hr at 37 C heat for 15 min to release antigens Note 100 L of the beads can be retained
21. as been selected by showing PATHATRIX P2 SELECTED Note To return to the default P1 run setting for processing food samples again press both arrow keys for 5 seconds then press the RIGHT arrow key to select P1 The display will then return to the standard PATHATRIX screen as normal Insert the Cartridge into the Pathatrix9 Auto Instrument until in the locking position Press the numbered button above the appropriate Cartridge The LED above the Cartridge will turn green indicating the run has begun P1 approximately 14 minutes P2 approximately 25 minutes Program P2 is used to process 10 mL environmental samples and is not AOAC validated If an AOAC validated protocol is desired environmental samples may be analyzed using P1 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Sample unloading 10 Chapter 1 Pathatrix Listeria spp Kit Linked to the DuPont BAX PCR System 1 Sample unloading At the end of the run the LED will flash red and green alternately Press the button above the appropriate Cartridge to initialize the draining step approximately 1 minute When the draining step is complete and the LED is illuminated red remove the Cartridge by pulling it out away from the instrument 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
22. consumable loading 1 Add PBS Cat no AM9624 diluted to 1X to the fill line of the Elution Vessel about 35 mL of 1X PBS 2 Place the lids on to the Sample and Elution Vessels making sure that the vessels are sealed all the way around 3 Ensure the beads are fully resuspended by agitating the bead vial e g vortexing bead vial or inversion of sealed vial and add 50 uL of the bead suspension beads from Cat no APL50 into the spout on the lid of the Sample Vessel Sample Vessel Spout Elution Vessel Sample Vessel Sterile 1X PBS 35 mL Sample Vessel Holder ATUBEHOLD 4 Remove the plastic 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 wwe P ww Capture Phase Valve Plunger 5 Holding both vessels lift the assembled vessels and attached kit out of the Sample Vessel Holder 22 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Chapter 2 Pathatrix Listeria spp Kit Linked to the Idaho Technology R A P I D LT PCR System 2 Samples and consumable loading 6 Place the vessels into the Cartridge pushing them in firmly from the bottom upwards 7 Next firmly push the remainder of the kit into the Cartridge ensuring that the valve the phase and the syringe are all held securely in the molded recess of the Cartridge 8 Push the magnet slider across into the locking posit
23. dled and disposed using safe laboratory practices in accordance and compliance with all appropriate regulations Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 19 Chapter 2 Pathatrix Listeria spp Kit Linked to the Idaho Technology R A P I D LT PCR System Sample enrichment Sample enrichment 20 Note Certain food types and swabs sponges which may have a very high background flora can benefit from an alternative enrichment strategy incorporating a second enrichment in MOPS BLEB see Appendix A Specific Recommendations for High Background Microflora 1 Food samples a Prepare a 1 in 10 dilution of the food sample in prewarmed 37 1 C Half Fraser broth Oxoid Product Code CM0895 with supplement Oxoid Product Code SRO166E in a sterile bag Note For example add 25 g of food sample to 225 mL of prewarmed media b Homogenize the sample by hand mixing for 10 15 seconds Massage the sample between the fingers to disperse any large clumps of material Narang and Cray 2006 Environmental contact swabs a Swab of an area with a rehydrated pre wetted swab and transfer the swab to a sterile bag Note Pre wetted swabs may be purchased from major laboratory suppliers or swabs may be pre wetted with 0 5 mL of Dey Engley Neutralizing Broth D E broth or enrichment media b Add 50 mL of pre warmed 37 1 C appropriately supplemented Half Fraser broth Oxoid CM0895 with supplement
24. e numbered button above the appropriate Cartridge to start the run The associated LED will turn green to indicate the run has started P1 approximately 14 minutes P2 approximately 25 minutes Program P2 is used to process 10 mL environmental samples and is not AOAC validated If an AOAC validated protocol is desired environmental samples may be analyzed using P1 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Chapter 2 Pathatrix Listeria spp Kit Linked to the Idaho Technology R A P I D LT PCR System Sample unloading Sample unloading 1 At the end of the run the LED will flash 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 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 in the Tube Rack also known as the Sample Vessel Holder or rack 6 Remove the lid from the Elution Vessel and leaving the Elution Vessel in place in the rack lift away the rest of the consumable including the Sample Vessel and discard 7 Place the Elution Vessel in the
25. ed materials from Life Technologies 200000 cece cece cece eee e eee e eens 40 APPENDIX D Safety orita 41 Chemicals teen tao e a Oda dd e a e N AS 41 Specificichemical handling sesei iera a ad 42 Biological hazard safety voii ir A A RA 42 Documentation and Support coccccccccc eee ees 43 Obtaining SDSS nuit a hates Sued hain ly auth stared bay wae tes Gaetan Seg 43 Obtaining Certificates of Analysis 0000 nnen ccc tte e ences 43 Obtaining Support 2 5 65 5 5 iiei ed ee eee de eee Biwi Pele bee eed e bee Pee ee pe ees 43 Limited product warranty x 2 205 40 6 pss sede gave a 43 Referente aora be 44 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA About the kit Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Product Information IMPORTANT Before using this product read and understand the information in Appendix D Safety on page 41 CAUTION Listeria monocytogenes and other Listeria spp are Biosafety Level 2 BSL 2 organisms Pregnant women or immuno compromised individuals in particular should understand the severe potential risks associated with working with Listeria monocytogenes Care must be taken when handling samples that may contain Listeria spp Laboratory personnel must be adequately trained to handle pathogens before being permitted to analyze samples for Listeria spp Laboratory personnel must wear appropriate protect
26. erile bag Note For example add 25 g of food sample to 225 mL of prewarmed media b Homogenize the sample by hand mixing for 10 15 seconds Massage the sample between the fingers to disperse any large clumps of material Narang and Cray 2006 Environmental contact swabs a Swab of an area with a rehydrated pre wetted swab and transfer the swab to a sterile bag Note Pre wetted swabs may be purchased from major laboratory suppliers or swabs may be pre wetted with 0 5 mL of Dey Engley Neutralizing Broth D E broth or enrichment media b Add 50 mL of pre warmed 37 1 C appropriately supplemented Half Fraser broth Oxoid CM0895 with supplement SRO166E or equivalent e g BLEB c Homogenize the sample by hand mixing for 10 15 seconds Massage the sample between the fingers to disperse any large clumps of material Narang and Cray 2006 Alternative For enrichment of environmental contact swabs sterile 15 mL tubes may be used as an alternative to sterile bags a Add 10 mL of pre warmed media to a sterile 15 mL tube b After swabbing an area twirl swab in the media in the 15 mL tube for approximately 1 minute Alternative Environmental contact sponges may be used as an alternative to swabs a Wipe of an area with a rehydrated pre wetted sponge and transfer the sponge to a sterile bag Note Sponges may be pre wetted with 10 mL of Dey Engley Neutralizing Broth D E broth or enrichment media b Add 10
27. est result interpretation and classification The Pathatrix Listeria spp Kit is designed as a sample preparation method for presence absence detection of Listeria spp in food matrices Using the Pathatrix Listeria spp Kit linked to VIDAS immunoassays presumptive results can be obtained prior to confirmation within 45 50 hours Once confirmed the results are reported as e Listeria spp Detected in 25 g sample matrices e Listeria spp Not detected in 25 g sample matrices Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 37 38 Specific Recommendations for High Background Microflora For certain food groups or environmental swabs particularly where high levels of background microflora are present the Listeria spp assay results can be enhanced by the use of a secondary MOPS BLEB broth after enrichment in Half Fraser The level of background contamination on the agar plates can be reduced by this method Examples of food groups that benefit from the use of this approach are Raw meat Raw shellfish Fresh fin fish Raw vegetables Leafy produce Salads Fruits Certain ready meals and Ready to eat RTE products with high levels of background microflora Contact swabs from highly contaminated areas Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Background Product overview Description of target microorganisms Audience Kit sensitivity Operating
28. for plating oR 500 pL Bead free supernatant Immediately atter heating capture Remove 500 uL of bead free Proceed with the assay in accordance beads for 1 min on the supernatant and inoculate the with the manufacturer s instructions DynaMag 2 Magnet VIDAS strip If a positive ELISA result is obtained an aliquot of the Pathatrix beads should be plated out see the following section Detection direct plating optional 36 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Chapter 3 Pathatrix Listeria spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System 3 Detection direct plating optional Detection direct plating optional Note We recommend streaking the sample out to generate individual colonies as opposed to spread plating 1 Streak 50 uL of unlysed Pathatrix bead suspension over well dried PALCAM and Listeria Oxford or equivalent agar plates e g Chromogenic Listeria agar ALOA 2 Allow the plates to dry for approximately 10 minutes then invert and incubate at 37 1 C for 18 48 hours as appropriate 24 48 hours was used in the AOAC RI validation study A presumptive positive result is defined as the isolation of typical suspicious or atypical Listeria spp colonies on the agar plates used A presumptive positive isolate should be subsequently confirmed by the use of subculture and appropriate biochemical and serological tests as required T
29. h supplement SRO166E or equivalent e g BLEB c Homogenize the sample by hand mixing for approximately 1 minute Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Chapter 1 Pathatrix Listeria spp Kit Linked to the DuPont BAX PCR System 1 Sample preparation 2 Incubate ALL samples at 37 1 C for a minimum of 24 hours Note We recommend that these sub samples for analysis are processed immediately or if storage is required that the samples are refrigerated at 5 3 C Samples should be rewarmed to 37 1 C prior to processing on the Pathatrix Auto Instrument The remaining enriched sample should be stored until the results have been determined Sample preparation 1 Remove the Sample and Elution Vessel from the consumable kit packaging and place into the Sample Vessel Holder 2 Loosen the lids from the vessels and partially remove leaving an opening through which to add sample and wash buffer 3 Food samples Prepare a 50 mL sample in the Sample Vessel by transferring a 50 mL aliquot from the enriched sample Swab samples Prepare a 10 mL sample in the Sample Vessel by transferring a 10 mL aliquot from the enriched sample Sponge samples Prepare a 10 mL sample in the Sample Vessel by transferring a 10 mL aliquot from the enriched sample Note If the samples are highly particulate and or contain a high fat content a Seward plain sterile bag with internal strainer may be used Seward
30. he samples are highly particulate and or contain a high fat content a Seward plain sterile bag with internal strainer may be used Seward Product Code BA6041 STR or equivalent Store the individual enriched samples at 5 3 C for potential reanalysis until the test result is confirmed Note Do not store for more than 32 hours If refrigerated after enrichment samples should be rewarmed to 37 1 C prior to removal of aliquots for analysis Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 31 3 Chapter 3 Pathatrix Listeria spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System Samples and consumable loading Samples and consumable loading 1 Add PBS Cat no AM9624 diluted to 1X to the fill line of the Elution Vessel about 35 mL of 1X PBS 2 Place the lids on to the Sample and Elution Vessels making sure that the vessels are sealed all the way around 3 Ensure the beads are fully resuspended by agitating the bead vial e g vortexing bead vial or inversion of sealed vial and add 50 uL of the bead suspension beads from Cat no APL50 into the spout on the lid of the Sample Vessel Sample Vessel Spout Elution Vessel Sample Vessel Sterile 1X PBS 35 mL Sample Vessel Holder ATUBEHOLD 4 Remove the plastic 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 wwe P ww Captu
31. ion and classification 0 00 cece cece eee eee eee ees 27 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 3 Contents HH CHAPTER 3 Pathatrix Listeria spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System 29 Workflow iva A dad 29 Procedural quidelin s cocinas tn ad RR toe 29 Sample eric ment e edo nt el e la hes Ok A O rd Ae 30 Sample preparation 000 c eee ee eee e EEEE 31 Samples and consumable loading 000cee cece cece eee eet tte eeeeeeeees 32 Sample nloadihg ssa A Aste dae e da sd lt att al des Se oh ee Ag 35 Detection VIDAS immunoassay testing protocol 0 ccc ccc cence ence eens 36 Detection direct plating optional 00 ccc ccc ence ne nee ne rnn 37 Test result interpretation and classification 00 000 eee eee eee eens 37 APPENDIX A Specific Recommendations for High Background Microflora cc cece eee e eee eee eeeeees 38 APPENDIX B Background is 0t08 c22 0a atada te wwe toh Gea 39 Product Over Wai A Gatley puke Late eg uh ae ta 39 Description of target microorganisMS 2 20 00 ccc eee eee eee ee eee eens 39 AUCIEN CE et atts ae ekg Wale dia tid ARA RARA 39 IIL SONSIEIVINY cvs e ele treat Moi ene a ec oats les Me eae a cere es 39 Operating conditions asiu s eee eae el eos a ae La enel ada es a 39 APPENDIX C Ordering Information 0ce cece eee eeee 40 Relat
32. ion 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 Phase Sliding Magnet Holder Magnet Release Button Valve Plunger Sample Vessel Elution Vessel Cartridge Holder ACARTHOLD 9 Reset the magnets into the locking position Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 23 24 Chapter 2 Pathatrix Listeria spp Kit Linked to the Idaho Technology R A P 1 D 9 LT PCR System Samples and consumable loading 10 Food samples Program 1 P1 is used for processing 50 mL food samples and P1 is the Default mode on the Pathatrix9 Auto Instrument Swab samples Program 2 P2 is used for processing 10 mL swab samples and can be selected by depressing the scroll arrow keys located either side of the LCD display simultaneously for approximately 5 seconds then pressing the left arrow key once to select P2 The display will now indicate that P2 has been selected by showing PATHATRIX P2 SELECTED Note To return to the default P1 run setting for processing food samples again press both arrow keys for 5 seconds then press the RIGHT arrow key to select P1 The display will then return to the standard PATHATRIX screen as normal Insert the Cartridge into the Pathatrix9 Auto Instrument until it clicks into the locking position Press th
33. ious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Documentation and Support Obtaining SDSs Safety Data Sheets SDSs are available from www lifetechnologies com support Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Obtaining Certificates of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Obtaining support Support email foodsafety lifetech com For the latest services and support information for all locations go to www lifetechnologies com support At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Search for user documents SDSs application notes formulations ha
34. itations may apply Specific chemical handling CAS Chemical Phrase 26628 22 8 Sodium Azide Sodium azide may react with lead and copper plumbing to form highly explosive metal azides Biological hazard safety 42 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 Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infect
35. ive equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves Waste should be disposed of in compliance with local and national legislation as appropriate The Pathatrix Listeria spp Kit provides a sample preparation method for presence absence tests based on the detection of as few as 1 10 cfu colony forming units 25 g of food sample An AOAC validated protocol for using this kit for sample preparation followed by detection of microorganisms by selective agar plates can be downloaded from www lifetechnologies com pathatrix Pathatrix Listeria spp Kit Linked to Selective Agar Plates Pub no MAN0007093 This user guide provides 3 alternative protocols for using the Pathatrix Listeria spp Kit followed by detection of microorganisms by PCR or ELISA Presumptive results can be obtained within the following times e 28 30 hours when linked to the DuPont BAX PCR system Chapter 1 e 25 27 hours when linked to the Idaho Technology R A PID 9 LT PCR system Chapter 2 e 45 50 hours when linked to the bioM rieux VIDAS immunoassay testing system Chapter 3 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 e Listeria spp Detected in 25 g sample matrices e Listeria spp Not detected in 25 g sample matrices See Ap
36. l for use with the Pathatrix Auto Instrument ACARTRACK holds 5 Cartridges DynaMag 2 Magnet for use with microcentrifuge tubes 123 21D Magnetic Capture Plate MAGNETICPLATE Reagents PBS 10X pH 7 4 AM9624 or AM9625 Related PCR assay MicroSEQ Listeria spp Detection Kit 4427410 40 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 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 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 Chemical safety AN 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 SD
37. ld be worn when weighing out powders e Care must be taken when boiling agar prior to autoclaving and heat resistant gloves should be worn when handling hot flasks of liquid e Take care when handling plates or vessels that contain microorganisms e Avoid generating aerosols as pathogenic organisms may be present e 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 e 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 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 29 Chapter 3 Pathatrix Listeria spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System Sample enrichment Sample enrichment 30 Note Certain food types and swabs sponges which may have a very high background flora can benefit from an alternative enrichment strategy incorporating a second enrichment in MOPS BLEB see Appendix A Specific Recommendations for High Background Microflora 1 Food samples a Prepare a 1 in 10 dilution of the food sample in prewarmed 37 1 C Half Fraser broth Oxoid Product Code CM0895 with supplement Oxoid Product Code SRO166E in a st
38. mask should be worn when weighing out powders e Care must be taken when boiling agar prior to autoclaving and heat resistant gloves should be worn when handling hot flasks of liquid e Take care when handling plates or tubes that contain microorganisms e Avoid generating aerosols as pathogenic organisms may be present e 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 e 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 Chapter 1 Pathatrix Listeria spp Kit Linked to the DuPont BAX PCR System Sample enrichment Sample enrichment 10 Note Certain food types and swabs sponges which may have a very high background flora can benefit from an alternative enrichment strategy incorporating a second enrichment in MOPS BLEB see Appendix A Specific Recommendations for High Background Microflora 1 Food samples a Prepare a 1 in 10 dilution of the food sample in prewarmed 37 1 C Half Fraser broth Oxoid Product Code CM0895 with supplement Oxoid Product Code SRO166E in a sterile bag Note For example add 25 g of food sample to 225 mL of prewarmed media b H
39. mately 1 minute When the draining step is complete and the LED is illuminated red remove the Cartridge by pulling it out away from the instrument 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 Place both vessels in the Tube Rack also known as the Sample Vessel Holder or rack Remove the lid from the Elution Vessel and leaving the Elution Vessel in place in the rack lift away the rest of the consumable including the Sample Vessel and discard Place the Elution Vessel in the rack and cap with a flat lid provided in the consumable kit Leave in the rack for 1 minute to allow capture of the beads Remove all the liquid from the Elution Vessel without removing the vessel from the rack taking care not to disturb the captured beads Remove the Elution Vessel from the vessel holder and use 700 uL of prewarmed BPW to resuspend the Pathatrix beads Transfer 600 uL to a microcentrifuge tube Note 100 uL of the Pathatrix beads can be retained for direct plating prior to the additional 2 hour growth step Vortex the tube for 10 seconds Incubate the tube for 2 hours at 37 1 C Remove the tube from the incubator and vortex for 10 seconds Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 35 2 Ch
40. ndbooks certificates of analysis citations and other product support documents e Obtain information about customer training 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 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 43 44 Reference Narang N and Cray W C 2006 Evaluation of Hand Mixing of Ground Beef and Poultry Samples as an Alternative to Stomaching for the Detection of Salmonella Food Protection Trends 26 14 19 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www lifetechnologies com support or email techsupportldlifetech com technologies www lifetechnologies com September 2012
41. omogenize the sample by hand mixing for 10 15 seconds Massage the sample between the fingers to disperse any large clumps of material Narang and Cray 2006 Environmental contact swabs a Swab of an area with a rehydrated pre wetted swab and transfer the swab to a sterile bag Note Pre wetted swabs may be purchased from major laboratory suppliers or swabs may be pre wetted with 0 5 mL of Dey Engley Neutralizing Broth D E broth or enrichment media b Add 50 mL of pre warmed 37 1 C appropriately supplemented Half Fraser broth Oxoid CM0895 with supplement SRO166E or equivalent e g BLEB c Homogenize the sample by hand mixing for 10 15 seconds Massage the sample between the fingers to disperse any large clumps of material Narang and Cray 2006 Alternative For enrichment of environmental contact swabs sterile 15 mL tubes may be used as an alternative to sterile bags a Add 10 mL of pre warmed media to a sterile 15 mL tube b After swabbing an area twirl swab in the media in the 15 mL tube for approximately 1 minute Alternative Environmental contact sponges may be used as an alternative to swabs a Wipe of an area with a rehydrated pre wetted sponge and transfer the sponge to a sterile bag Note Sponges may be pre wetted with 10 mL of Dey Engley Neutralizing Broth D E broth or enrichment media b Add 100 mL of pre warmed 37 1 C appropriately supplemented Half Fraser broth Oxoid CM0895 wit
42. package insert instructions and instrument user manual for information regarding the correct set up and operation of the BAX PCR system 1 Pipette 25 uL of the Pathatrix bead suspension into BAX lysis tubes and proceed according to standard BAX lysis procedure 2 Once the lysis step is complete immediately place the Rack and Tubes onto the Magnetic Capture Plate Cat no MAGNETICPLATE and leave for at least 1 minute to allow the Pathatrix beads to accumulate on the bottom of the tube 3 Transfer only bead free lysate into the BAX PCR reaction tubes Note Target DNA if present will be in the bead free supernatant 4 To proceed refer to the DuPont Qualicon operating instructions Standard sample L 25 uL Add 100 pL PBS to resuspend Transfer 25 uL of bead suspension into BAX lysis Tubes containing standard lysis reagents including protease the beads Bead free lysate Magnetic plate Perform standard BAX lysis procedure Magnetic plate Capture beads for 1 min Transfer bead free lysate into BAX PCR Reaction Tubes If a positive PCR result is obtained an aliquot of the Pathatrix beads should be plated out see the following section Detection direct plating optional 16 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Chapter 1 Pathatrix Listeria spp Kit Linked to the DuPont BAX P
43. pendix B on page 39 for additional background information Product Information Kit contents Kit contents The Pathatrix Listeria spp Kit Cat no APL50 contains enough consumable components and Pathatrix beads to process 50 samples Item tant ye Storage volume Pre sterilized Sample and Elution 50 each Room temperature Vessel Packs Pre sterilized Capture Phase Packs 50 each Room temperature Pre sterilized Flat Cap Lids 50 each Room temperature Anti Listeria spp Antibody Coated 2 5 mL 50 tests 5 3 C Paramagnetic Beads t The beads have a shelf life of 12 months and are labeled with an expiration date accordingly IMPORTANT Never freeze the Pathatrix bead suspension Beads that have been subjected to freezing temperatures may be rendered inactive Note Parts may ship separately depending on configuration and storage conditions Materials not included in the kit The following table includes materials and equipment for using but not included in the Pathatrix consumables kits Unless otherwise indicated many of the listed items are available from major laboratory suppliers MLS Equipment consumables and reagents Item Source Equipment Incubator 37 1 C MLS Pathatrix Auto Instrument Life Technologies Cat no PATHATRIXAUTO For use with the BAX PCR protocol Magnetic Capture Plate Life Technologies Cat no MAGNETICPLATE For use with the R A P I D L
44. re Phase Valve Plunger 5 Holding both vessels lift the assembled vessels and attached kit out of the rack also known as the Sample Vessel Holder 32 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Chapter 3 Pathatrix Listeria spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System 3 Samples and consumable loading 6 Place the vessels into the Cartridge pushing them in firmly from the bottom upwards 7 Next firmly push the remainder of the kit into the Cartridge ensuring that the valve the phase and the syringe are all held securely in the molded recess of the Cartridge 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 Phase Sliding Magnet Holder Magnet Release Button Valve Plunger Sample Vessel Elution Vessel Cartridge Holder ACARTHOLD 9 Reset the magnets into the locking position Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA 33 34 Chapter 3 Pathatrix Listeria spp Kit Linked to the bioM rieux VIDAS Immunoassay Testing System Samples and consumable loading 10 Food samples Program 1 P1 is used for processing 50 mL food samples and P1 is the Default mode on the Pathatrix9 Auto Instrument Swab samples Program 2 P
45. sults 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 or to use the product to perform other tests on a contract or fee per test basis for or on behalf of third parties For information on obtaining additional rights please contact outlicensing dlifetech com TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners in the United States and other countries BAX is a registered trademark DuPont or its affiliates R A P I D is a registered trademark of Idaho Technology Inc Stomacher is a registered trademark of Seward Limited UK VIDAS is a registered Trademark of bioM rieux Whirl Pak is a registered trademark of Wheatherby Nasco Inc 2012 Life Technologies Corporation All rights reserved Contents Product Information xiii aaa 5 AbOUtihe kit sat a ea SR Se ee ee reas 5 Wi COMLGIIIS 2 Ae cate h
46. x Listeria spp Kit Individual Samples Linked to PCR or ELISA 17 1 Chapter 1 Pathatrix Listeria spp Kit Linked to the DuPont BAX PCR System Test result interpretation and classification 18 Pathatrix Listeria spp Kit Individual Samples Linked to PCR or ELISA Workflow Pathatrix Listeria spp Kit Linked to the Idaho Technology R A P 1 D 9 LT PCR System Sample enrichment page 20 h Sample preparation page 21 h Samples and consumable loading page 22 vy Sample unloading page 25 v Detection R A P 1 D 9 LT PCR protocol page 26 h Detection direct plating optional page 27 vy Test result interpretation and classification page 27 Procedural guidelines e Use aseptic technique and good laboratory practices at all times e A facemask should be worn when weighing out powders e Care must be taken when boiling agar prior to autoclaving and heat resistant gloves should be worn when handling hot flasks of liquid e Take care when handling plates or vessels that contain microorganisms e Avoid generating aerosols as pathogenic organisms may be present e 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 e Sample enrichments might be contaminated with pathogenic organisms infectious to humans so all waste must be treated as biohazardous and han
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