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PMA-PCRkitsaredesignedforselectivedetectionofviablebacteriafromaspecificstrainusingPMAdyeandreal-timePCR.ThekitscontainPMAdye,Forget-Me-Not™qPCRMasterMix,andPCRprimersfordetectionofselectedstrainsofbacteriathatareofwidespreadinteresttofoodsafety,publichealth,and/orantibacterialresearch.
ThiskitcontainsprimersforamplificationofMycobacteriumtuberculosis groEL2gene,withreagentssufficienttotreat80bacterialcultureswithPMAandperform200PCRreactions.ThenumberofsamplesthatcanbetreatedwithPMAusingthekitmayvarydependingonsampletype.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.
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.
Mycobacteriumtuberculosisisapathogenicbacteriathatinfectsthelungsandcausesthediseasetuberculosis.PCRtodetectMycobacteriumtuberculosishasbeenreportedusingtheprimersprovidedinthekit (seeReference2underthereferencestab).Inaddition,theseprimershavebeenvalidatedatBiotiumforreal-timeqPCRusingForget-Me-Not™MasterMix(seeproductprotocolunderdownloadsfordetails). Note:groEL2primersalsoamplifyothermycobacteriaspecies(seeReference2underthereferencestab),butproductsmaybedistinguishablebymeltcurveanalysis.
AlsoseeourotherPMA-PCRkitsfordetectionofE.coli,E.colistrain0157:H7,Staphylococcusaureus,methicillin-resistantStaphylococcusaureus(MRSA),Listeriamonocytogenes,Legionellapneumophila,andSalmonellaenterica.Don’tseeyourfavoritestrain?Letusknowattechsupport@biotium.com.
MaterialsfromBiotiumaresoldforresearchuseonly.
1.NockerA,etal.Comparisonofpropidiummonoazidewithethidiummonoazidefordifferentiationoflivevs.deadbacteriabyselectiveremovalofDNAfromdeadcells.J.Microbiol.Meth.67(2),310-320(2006).
2.PaoCC, etal.DetectionandidentificationofMycobacteriumtuberculosisbyDNAamplification.J.Clin.Microbiol.28(9),1877-80(2006).
3.FittipaldiM,etal.ProgressinunderstandingpreferentialdetectionoflivecellsusingviabilitydyesincombinationwithDNAamplification.J.Microbiol.Meth.91(2),276-289(2012).
DownloadthePMAreferencelist
SouthernBiotech/Goat Anti-Human Lambda-Alexa Fluor® 555/2070-32/1.0 mg
vectorlabs/Biotinylated Aleuria Aurantia Lectin (AAL)/B-1395/1 mg
vectorlabs/VECTASTAIN® Elite® ABC-HRP Kit (Peroxidase, Mouse IgG)/PK-6102/1 kit
vectorlabs/VECTASTAIN® Elite® ABC-HRP Kit (Peroxidase, Universal)/PK-6200/1 kit
vectorlabs/Unconjugated Musa Paradisiaca (Banana) Lectin (BanLec)/L-1410/5 mg
GiottoBiotech/Calmodulin N60D/5 mg/G02CLM60cn