PMA-PCRkitsaredesignedforselectivedetectionofviablebacteriafromaspecificstrainusingPMAorPMAxx™dyeandreal-timePCR.ThekitscontainPMAorPMAxx™dye,Forget-Me-Not™qPCRMasterMix,andPCRprimersfordetectionofselectedstrainsofbacteriathatareofwidespreadinteresttofoodsafety,publichealth,and/orantibacterialresearch.
ThiskitcontainsprimersforamplificationofE.coli strainO157:H7Z3276gene,withreagentssufficienttotreat80bacterialcultureswithPMAorPMAxx™,andperform200PCRreactions.ThereisanoptiontochooseeitherPMAorPMAxx™fortheviABIlitydye(seebelowformoreinformationaboutthesedyes).ThenumberofsamplesthatcanbetreatedwithPMAorPMAxx™usingthekitmayvarydependingonsampletype.Seetheproductprotocolunderthedownloadstabandreferencesformoreinformation.
Kitcontents:
- PMAdyeORPMAxx™dye,20mMinwater,100uL
- 5XPMAEnhancerforGramNegativeBacteria,16mL
- 2XForget-Me-Not™qPCRMasterMix,2x1mL(200reactions)
- ROXreferencedye,1mL
- Z3276primermix,5uM,400uL
PMAReal-TimePCRBacterialViabilityKits
| ProductName | SKU | BacteriaStrain | GeneName | Size | Purchase |
|---|---|---|---|---|---|
| PMAReal-TimePCRBacterialViabilityKit–E.coli(uidA) | 31050 | E.coli | uidA | 200assays | Purchase31050 |
| PMAReal-TimePCRBacterialViabilityKit–E.coliO157:H7(Z3276) | 31037 | E.coliO157:H7 | Z3276 | 200assays | Purchase31037 |
| PMAReal-TimePCRBacterialViabilityKit–Listeriamonocytogenes(hly) | 31051 | Listeriamonocytogenes | hly | 200assays | Purchase31051 |
| PMAReal-TimePCRBacterialViabilityKit–Salmonellaenterica(invA) | 31033 | Salmonellaenterica | invA | 200assays | Purchase31033 |
| PMAReal-TimePCRBacterialViabilityKit–E.coli(uidA) | 31050-X | E.coli | uidA | 200assays | Purchase31050-X |
| PMAReal-TimePCRBacterialViabilityKit–E.coliO157:H7(Z3276) | 31037-X | E.coliO157:H7 | Z3276 | 200assays | Purchase31037-X |
| PMAReal-TimePCRBacterialViabilityKit–Listeriamonocytogenes(hly) | 31051-X | Listeriamonocytogenes | hly | 200assays | Purchase31051-X |
| PMAReal-TimePCRBacterialViabilityKit–Salmonellaenterica(invA) | 31033-X | Salmonellaenterica | invA | 200assays | Purchase31033-X |
| PMAReal-TimePCRBacterialViabilityKit–Legionellapneumophila(mip) | 31053 | Legionellapneumophila | mip | 200assays | Purchase31053 |
| PMAReal-TimePCRBacterialViabilityKit–Mycobacteriumtuberculosis(groEL2) | 31034 | Mycobacteriumtuberculosis | groEL2 | 200assays | Purchase31034 |
| PMAReal-TimePCRBacterialViabilityKit–Staphylococcusaureus(mecA) | 31036 | Staphylococcusaureus | mecA | 200assays | Purchase31036 |
| PMAReal-TimePCRBacterialViabilityKit–Staphylococcusaureus(nuc) | 31035 | Staphylococcusaureus | nuc | 200assays | Purchase31035 |
PMAisahighaffinityphotoreactiveDNAbindingdyedevelopedbyBiotium.Thedyeisweaklyfluorescentbyitselfbutbecomeshighlyfluorescentuponbindingtonucleicacids.ItpreferentiallybindstodsDNAwithhighaffinity.Uponphotolysis,thephotoreactiveazidogrouponthedyeisconvertedtoahighlyreactivenitrenerADIcal,whichreadilyreactswithanyhydrocarbonmoietyatthebindingsitetoformastablecovalentnitrogen-carbonbond,thusresultinginpermanentDNAmodification.Thedyeiscellmembrane-impermeableandthuscanbeusedtoselectivelymodifyDNAfromdeadcellswithcompromisedmembraneintegrity,whileleavingDNAfromviablecellsintact.PMAinhibitsPCRamplificationofmodifiedDNAtemplatesbyacombinationofremovalofmodifiedDNAduringpurificationandinhibitionoftemplateamplificationbyDNApolymerases(seeReference1underthereferencestab).Consequentlythedyeisusefulintheselectivedetectionofviablepathogeniccellsbyquantitativereal-timePCR.
PMAxx™isanewandimprovedversionofPMAdesignedbyBiotiumscientiststobeasuperioralternativetoPMA.WhilePMAisgenerallyeffectiveatdifferentiatingbetweenliveanddeadbacteriabyqPCR,itdoesnotcompletelyeliminatePCRproductsfromdeadcell DNA.Thiscouldpotentiallygivefalsepositiveresults.Biotium’snewdyePMAxx™ismuchmoreeffectiveateliminatingPCRamplificationofdeadcellDNA,andthereforeprovidesthebest discriminationbetweenliveanddeadbacteria.
Forget-Me-Not™qPCRMasterMixisahot-startEvaGreen®dye-basedmastermixforuseinrealtimePCRapplicationsandDNAmeltcurveanalysis.Forget-Me-Not™mastermixcontainsalowconcentrationofbluedyewhichallowsyoutoseeataglancewhetheryouforgottoaddmastermixtoanyofyourtubes,soyoucancatchpipettingmistakesandavoidwastingtime,reagents,andyourpreciousDNAsamples.ItisformulatedforqPCRusingafastcyclingprotocol,butalsocanbeusedforqPCRusingregularcyclingprotocols.EvaGreen®dyeisauniqueDNA-bindingdyewithfeaturesidealforbothqPCRandmeltcurveanalysis.EvaGreen®dyebindstodsDNAviaanovel“release-on-demand”mechanism,whichpermitstheuseofarelativelyhighdyeconcentrationinqPCRwithoutPCRinhibition.Forget-Me-Not™MasterMixcontainsCheetah™Taq,Biotium’sfast-activatingchemically-modifiedhot-startTaqpolymerase,whichisparticularlysuitableforfastPCRcyclingprotocols.
E.coliO157:H7isapathogenicstrainofEscherichiacolibacteria.Oftenafood-bornepathogen,itcancausedigestiveillness. PMA-basedviabilityPCRtodetectE.coliO157:H7hasbeenreportedusingtheprimersprovidedinthekit,andORFZ3276wasshowntobespecificforstrainO157:H7 (seeReference2underthereferencestab).Forpan-E.coliprimerstodetectotherstrainsofE.colibesidesO157:H7,pleaseseekit31050.Inaddition,theseprimershavebeenvalidatedatBiotiumforreal-timeqPCRusingForget-Me-Not™MasterMix(seeproductprotocolunderdownloadsfordetails).
AlsoseeourotherPMA-PCRkitsfordetectionofListeriamonocytogenes, Mycobacteriumtuberculosis,Staphylococcusaureus,methicillin-resistantStaphylococcusaureus(MRSA),Legionellapneumophila,andSalmonellaenterica.Don’tseeyourfavoritestrain?Letusknowattechsupport@biotium.com.
MaterialsfromBiotiumaresoldforresearchuseonly.
References
1.NockerA,etal.Comparisonofpropidiummonoazidewithethidiummonoazidefordifferentiationoflivevs.deadbacteriabyselectiveremovalofDNAfromdeadcells.J.Microbiol.Meth.67(2),310-320(2006).
2.LiandChen.Real-timePCRmethodologyforselectivedetectionofviableEscherichiacoliO157:H7cellsbytargetingZ3276asageneticMarker.Appl.Environ.Microbiol.78(15),5297-304(2012).
3.FittipaldiM,etal.ProgressinunderstandingpreferentialdetectionoflivecellsusingviabilitydyesincombinationwithDNAamplification.J.Microbiol.Meth.91(2),276-289(2012).
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