Thermo Fisher Scientific Pathatrix 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates User guide

Brand: Thermo Fisher Scientific

Model: Pathatrix 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Type: User guide

USER GUIDE

For testing of Food and Environmental samples only.

Pathatrix® 5-Pooling Listeria spp. Kit Linked to

Selective Agar Plates

For use with the Pathatrix® Auto Instrument

Catalog Number APL250P

Publication Number MAN0006909

Revision 14 August 2012

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 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, 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 outlicensi[email protected]om.

TRADEMARKS

The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners in the United States and other countries.

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

UK. Whirl-pak is a registered trademark of Wheatherby/Nasco, Inc.

Contents

Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Product Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

About the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Kit contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Materials not included in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

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

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Sample pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Samples and consumable loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Sample unloading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Detection—direct plating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Retesting of individual samples after “Positive Pooled Sample” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Test result interpretation and classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

■APPENDIX A Specific Recommendations for

High-Background Microflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

■APPENDIX B Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Description of target microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Audience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Sampling protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Kit sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Operating conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

AOAC Performance Tested Methodssm Certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

■APPENDIX C Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Related materials from Life Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

4Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Contents

■APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Specific chemical handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Obtaining Certificates of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Product Information

IMPORTANT! Beforeusingthisproduct,readandunderstandtheinformationin

AppendixD,“Safety”onpage21.

WARNING! 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protective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.

About the kit

ThePathatrix®5‐PoolingListeriaspp.Kitprovidesasamplepreparationmethodfor

presence/absencetestingbasedonthedetectionofasfewas1–10cfu(colonyforming

units)/25goffoodsample.

UsingthePathatrix®5‐PoolingListeriaspp.KitLinkedtoSelectiveAgarPlates,

presumptiveresultscanbeobtainedwithin42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:

•Listeriaspp.Detectedin25g(samplematrices)

•Listeriaspp.Notdetectedin25g(samplematrices)

SeeAppendixBonpage18foradditionalbackgroundinformation.

6Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Product Information

Kit contents

ThePathatrix®5‐PoolingListeriaspp.Kit(Cat.no.APL250P)containsenough

consumablecomponentsandPathatrix®beadstoprocess250 samples(50 Cartridge

runs).

IMPORTANT! NeverfreezethePathatrix®beads.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).

Item Quantity or

volume Storage

Pre-sterilized Sample and Elution

Vessel Packs

50 each Room temperature

Pre-sterilized Capture Phase Packs 50 each Room temperature

Pre-sterilized Flat Cap Lids 50 each Room temperature

Anti-Listeria spp. Antibody-Coated

Paramagnetic Beads†

† The beads have a shelf life of 12 months and are labeled with an expiration date accordingly.

2.5 mL (50 tests) 5 ±3°C

Equipment, consumables, and reagents

Item Source

Equipment

Incubator, 37 ±1°C MLS

Pathatrix® Auto Instrument Life Technologies Cat. no. PATHATRIXAUTO

Consumables

Optional for environmental samples:

Swabs or sponges MLS

Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Product Information

Materials not included in the kit

Sterile bags for enrichment (Whirl-Pak® or

Stomacher® bag, or equivalent)

Nasco # B01196WA, Seward product code

BA6041, or equivalent

Optional for high-particulate or high-fat-

content samples:

• Sterile filter bags for enrichment (Whirl-

Pak® or Stomacher® bag, or equivalent)

•Pathatrix

® Foam filters

•Pathatrix

® 5-Pool Kit—Straws (254 mm)

and Syringes (10 mL)

• Nasco # B01348WA, Seward product code

BA6041/STR, or equivalent

• Life Technologies Cat. no. PFF

• Life Technologies Cat. no. POOL510MLN

Microcentrifuge tubes, PCR clean, 1.5-mL MLS

Sterile, disposable 10-µL loops MLS

Media

See page 10 and Appendix A on page 17 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.

Half Fraser broth

Half Fraser broth supplement

Oxoid product code CM0895

Oxoid product code SR0166E

Alternative for environmental samples:

•Buffered Listeria enrichment broth

(BLEB)

• BLEB supplement

• Oxoid product code CM0897

• Oxoid product code SR0141E

For optional secondary enrichment:

MOPS-BLEB Oxoid product code CM1071

Selective agar

PALCAM agar base

PALCAM selective supplement

Oxoid product code CM0877

Oxoid product code SR0150

Listeria selective agar (Oxford formulation)

Listeria selective supplement (Oxford

formulation)

Modified Listeria selective supplement

(Oxford formulation)

Oxoid product code CM0856

Oxoid product code SR0140

Oxoid product code SR0206

Alternative chromogenic agar:

•Brilliance Listeria agar base

•Brilliance Listeria selective supplement

• Oxoid product code CM1080

• Oxoid product code SR0227

Reagents

PBS, 10X, pH 7.4 Life Technologies Cat. no. AM9624 or AM9625

Equipment, consumables, and reagents

Item Source

8Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Product Information

Materials not included in the kit

Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Methods

Workflow

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tubesthatcontainmicroorganisms.

•Avoid

generatingaerosols,aspathogenicorganismsmaybepresent.

•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 (page 10)

Sample pooling (page 11)

Samples and consumable loading (page 12)

Sample unloading (page 15)

Detection—direct plating (page 16)

Retesting of individual samples after “Positive Pooled Sample” (page 16)

Test result interpretation and classification (page 16)

10 Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Methods

Sample preparation

Note: Certainfoodtypesandswabs/sponges,whichmayhave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

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

SR0166E)inasterilebag.

Note: Forexample,add25goffoodsampleto225mLofprewarmedmedia.

Environmentalcontactswabs

a. Prepareswabsforenrichment.Oncecontactswabbingofanarea

10cmx10cmhasbeencompletedwiththerehydrated,pre‐wettedswab,

transfertheswabtoasterilebag.

b. Add50mLofprewarmed(37±1°C)appropriatelysupplementedHalfFraser

broth(OxoidCM0895withsupplementSR0166Eorequivalent,e.g., BLEB).

2. 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).

3. IncubateALLsamplesat37 ±1°Cforaminimumof24 hours.

Note: Werecommendthatthesesub‐samplesforpooling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at5±3°Cforupto

32 hoursuntiltheresultsofthepooledsamplehavebeendetermined.

Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Methods

Sample pooling

1. RemovetheSampleandElutionVesselsfromtheconsumablekitpackagingand

placeintotheTubeRack(alsoknownastheSampleVesselHolder).

2. 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.

3. Foodsamples

Prepareasingle,pooledsampleintheSampleVesselbypooling5×10‐mL

aliquotsfromfiveindividuallyenrichedsamplestocreatea50‐mLpooledsample

(seethefollowingfigure).

Swabsamples

Prepareasingle,pooledsampleintheSampleVesselbypooling5×2‐mLaliquots

fromfiveindividuallyenrichedsamplestocreatea10‐mLpooledsample.

Note: Ifthesamplesarehighlyparticulateand/orcontainahighfatcontent,the

useoftheFiltaFoamsystem(Foamfilters,Cat.no.PFF)withpoolingsyringesand

straws(Cat.no.POOL510MLN)isrecommended.Alternatively,Sewardplain

sterilebagswithinternalstrainersmaybeused(Seward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 pooled sample is negative, then

all 5 individual samples are negative. If pooled sample is positive, then

retest all 5 individual samples postenrichment.

5X Individual

Samples

Individual

Enrichment of

Samples

Postenriched

Samples

Pooling of Samples

(5X 10-mL Samples)

50-mL Pooled Sample

(1X Pathatrix® Test)

12 Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Methods

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μLofthebeadsuspension(beads

fromCat. no.APL250P)intothespoutonthelidoftheSampleVessel.

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.

5. Holdingbothvessels,lifttheassembledvesselsandattachedkitoutoftherack

(alsoknownastheSampleVesselHolder).

Elution Vessel

Sterile 1X PBS (35 mL)

Sample Vessel Holder

(ATUBEHOLD)

Sample Vessel Spout

Sample Vessel

Capture Phase

Valve Plunger

Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Methods

Samples and consumable loading

6. PlacethevesselsintotheCartridge,pushingtheminfirmlyfromthebottom

upwards.

7. Next,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.

9. Resetthemagnetsintothelockingposition.

Syringe

Magnet Release Button

Valve Plunger

Phase

Sample Vessel

Elution Vessel

Cartridge Holder

(ACARTHOLD)

Sliding Magnet

Holder

14 Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Methods

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Pathatrix®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analyzing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.

11. InserttheCartridgeintothePathatrix®AutoInstrumentuntilinthelocking

position.

12. PresstheRUNbutton(i.e.,thenumberedbutton)abovetheappropriate

Cartridge.TheLEDabovetheCartridgewillturngreenindicatingtherunhas

begun.

†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® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Methods

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on

therack,liftawaytherestoftheconsumable,includingtheSampleVessel,and

discard.

7. Placeaspareflatlid(providedintheconsumablekit)ontopoftheElutionVessel

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. Asepticallyadd100μLofsterile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AWAYfrommagnets(e.g.,Pathatrix®vesselholders)at5±3°Cforup

to24hours.

16 Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Methods

Detection—direct plating

Note: Werecommendstreakingthesampleouttogenerateindividualcolonies,as

opposedtospreadplating.

1. Pipetteall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‐μL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).

Ifanegativeresultisobtainedfromthepooledsample,theindividualovernight

enrichmentscanbediscarded,asfurthertestingisnotrequired.

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,appropriatebiochemical

andserologicaltestsasrequired.

Ifapositiveresultisobtainedfromthepooledsample,theovernightenrichmentscan

beretestedtoallowidentificationofwhichindividualsamplesinthepoolproduced

thepositiveresult(seenextsection,Retestingofindividualsamplesafter“Positive

PooledSample”).

Retesting of individual samples after “Positive Pooled Sample”

1. Individualsamples,whichrequireretesting,shouldberewarmedto37±1°C

beforehaving10mLremovedandtransferredintoaSampleVessel.

2. Individualsamplesshouldberetestedimmediatelybyrepeatingallthestepsin

SamplesandconsumableloadingthroughDetection—directplatingdescribed

above.

Test result interpretation and classification

ThePathatrix®5‐PoolingListeriaspp.Kitisdesignedasasamplepreparationmethod

forpresence/absencedetectionofListeriaspp.infoodmatrices

UsingthePathatrix®5‐PoolingListeriaspp.KitLinkedtoSelectiveAgarPlates,

presumptiveresultscanbeobtained,priortoconfirmation,within42hours.

Onceconfirmed,theresultsarereportedas:

•Listeriaspp.Detectedin25g(samplematrices)

•Listeriaspp.Notdetectedin25g(samplematrices)

Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

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broth.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

18 Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Background

Product overview

Description of

target

microorganisms

ThegenusListeriaiscomposedofsixspecies;L.monocytogenes,L.innocua,L. welshimeri,

L.seeligeri,L.ivanovii,L.grayiand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.

Audience ThePathatrix®5‐Pooling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.

Sampling protocol ThestandardfoodsamplesizeusedinthePathatrix®Autosystemis25goffood

dilutedwith225mLofenrichmentmedium.Werecommendthatsub‐samplesfor

pooling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

withthePathatrix®Autosystem.Theremainingenrichedsampleshouldbestoredat

5±3°Cforupto32hoursuntiltheresultsofthepooledsamplehavebeendetermined.

Kit sensitivity Thesamplepreparationprocedureallowsyoutodetectasfewas1–10colony‐forming

units(cfu)from25gramsoffoodorfromenvironmentalswabandspongesamples

afterenrichment.ThelimitationofthePathatrix®5‐PoolingListeriaspp.Kitisinthe

abilityofthetargettoreproduceintheenrichmentmedium,becapturedbythe

magnet,andsubsequentlybeisolatedonselectiveagarplates.

CAUTION! ThePathatrix®kithasbeenevaluatedonsoftcheese,cream,andcooked

chickenfoodmatrices.Giventhewidevarietyofproductsandmanufacturing

procedures,itisrecommended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.

Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Appendix B Background

AOAC Performance Tested Methodssm Certification B

Operating

conditions

ThePathatrix®AutoInstrumentisforindooruseonlyandforaltitudesnotexceeding

2000m(6500ft.)abovesealevel.

AOAC Performance Tested Methodssm Certification

ThePathatrix®5‐PoolingListeriaspp.Kithasbeenvalidatedandcertifiedbythe

AOACResearchInstitute(Licensenumber090201B)forthedetectionofListeriaspp.in

foodmatrices,includingsoftcheese,cream,andcookedchicken.Thetestoperatesin

conjunctionwithapooledPathatrix®workflow.Astandard25‐gfoodsampleishand

mixedbrieflywith225mLofenrichmentmediumandincubatedforaminimumof

24 hoursat37°C.Afterincubation,a10‐mLaliquotofthesampleiscombinedwithup

to4additional10‐mLsamplealiquots,andtheresulting50‐mLpooledsampleisthen

subjectedtoa15‐minutePathatrix®AutoInstrumentcapturecycle.

Themethodwasvalidatedforusewiththefollowingmatrices:

•25gofsoftcheese

•25gofcream

•25gofcookedchicken

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%

20 Pathatrix® 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Ordering Information

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Equipment

Pathatrix® Auto Instrument PATHATRIXAUTO

Cartridge Rack (optional for use with the Pathatrix® Auto Instrument;

holds 5 Cartridges)

ACARTRACK

DynaMag™-2 Magnet (for use with microcentrifuge tubes) 123.21D

Consumables

Foam filters PFF

5-Pool Kit—Straws (254 mm) and Syringes (10 mL) POOL510MLN

Reagents

PBS, 10X, pH 7.4 AM9624 or AM9625

Related PCR assay

MicroSEQ® Listeria spp. Detection Kit 4427410

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Thermo Fisher Scientific Pathatrix 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates User guide

Brand: Thermo Fisher Scientific

Model: Pathatrix 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates

Type: User guide

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Thermo Fisher Scientific Pathatrix 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates User guide

Thermo Fisher Scientific Pathatrix 5-Pooling Listeria spp. Kit Linked to Selective Agar Plates User guide

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