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:
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.
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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Biotium公司是一家在荧光技术领域独树一帜的生物技术公司,公司位于美国加利福尼亚的BAY AREA------世界上首要的生物科技中心。该公司一直致力于开发和生产用于生物医学研究的荧光指示剂及其他特殊生化产品。其产品被广泛应用于细胞生物学、蛋白质组学、分子生物学、免疫学、微生物学、诊断学、生物化学、神经科学、血液流动检测及大规模药物筛选等众多领域。Biotium公司专利产品众多,包括已经被广泛使用的实时PCR定量荧光染料EvaGreen、具有划时代意义的凝胶核酸染料GelRed和GelGreen,以及最新推出的Cheetah Taq热启动酶个CF系列蛋白质标记染料等等。