PMA-PCRkitsaredesignedforselectivedetectionofviablebacteriafromaspecificstrainusingPMAdyeandreal-timePCR.ThekitscontainPMAdye,Forget-Me-Not™ qPCRMasterMix,andPCRprimersfordetectionofselectedstrainsofbacteriathatareofwidespreadinteresttofoodsafety,publichealth,and/orantibacterialresearch.
ThiskitcontainsprimersforamplificationofLegionellapneumophila mipgene,withreagentssufficienttotreat80bacterialcultureswithPMAandperform200PCRreactions.ThenumberofsamplesthatcanbetreatedwithPMAusingthekitmayvarydependingonsampletype.Seetheproductprotocolunderthedownloadstabandreferencesformoreinformation.
Kitcontents:
- PMAdye,20mMinwater,100uL
- 5XPMAEnhancerforGramNegativeBacteria,16mL
- 2XForget-Me-Not™ qPCRMasterMix,2x1mL(200reactions)
- ROXreferencedye,1mL
- mipprimermix,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.
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.
Legionellapneumophilaisapathogenicspeciesofgram-negativebacteria.ItcaninfectthelungsandcauseLegionnaire’sDisease.Legionellapneumophilaissometimesdetected inwater sourcessuchascoolingtowersandswimmingpools.PCRtoamplifythegenemiphasbeenpublishedandishighlyspecificforLegionella (seeReference2underthereferencestab).TheprimersprovidedinthekithavebeenvalidatedatBiotiumforreal-timeqPCRusingForget-Me-Not™MasterMix(seeproductprotocolunderdownloadsfordetails).
AlsoseeourotherPMA-PCRkitsfordetectionofEscherichiacoli, E.coli O157:H7, Mycobacteriumtuberculosis,Staphylococcusaureus,methicillin-resistantStaphylococcusaureus(MRSA),Listeriamonocytogenes andSalmonellaenterica.Don’tseeyourfavoritestrain?Letusknowattechsupport@biotium.com.
MaterialsfromBiotiumaresoldforresearchuseonly.
References
1.Nocker,A.,etal.Comparisonofpropidiummonoazidewithethidiummonoazidefordifferentiationoflivevs.deadbacteriabyselectiveremovalofDNAfromdeadcells.J.Microbiol.Meth.67(2),310-320(2006).
2.WilsonDA,etal.DetectionofLegionellapneumophilabyreal-timePCRforthemipgene.J.Clin.Microbiol.41(7)3327-3330(2003).
3.Fittipaldi,M.,etal.ProgressinunderstandingpreferentialdetectionoflivecellsusingviabilitydyesincombinationwithDNAamplification.J.Microbiol.Meth.91(2),276-289(2012).
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