商品介绍
MycoDect™MycoplasmaDetectionKit:#MD050
ProductDescription
Mycoplasmasaresmall,roundorfilamentousprokaryoticorganismsthatlackcellwalls.Ithasbeenestimatedthatatleast15%-35%cellculturesarecontaminatedwithmycoplasma.Mycoplasmacontaminationcanmodifymanyaspectsofcellphysiologywithoutcausingobviousmediumcloudinessandapparenteffectoncellgrowth.Mycoplasmacontaminationaffectsupto80%ofcontinuouscellcultures.Ifundetected,mycoplasmacontaminationcanhavesignificanteffectsonthequalityandreliABIlityofyourcellculturepreparations.Duetotheirsmallsizeanddeformability,mycoplasmascanpassthrough0.22µmfilter.Thelackofcellwallmakesmycoplasmaunresponsivetocommonantibioticsthattargetcellwallsynthesis.Itisessentialthatallcellstocksandnewculturesenteringafacilityaretestedforthepresenceofmycoplasmaandallcellculturesshouldbetestedroutinely(e.g.onceevery2-3months)inordertomaintainamycoplasma-freeenvironment.
Manymethodsareavailablefordetectionofmycoplasma,includingisolationinbroth/agarculture,directorindirectfluorescencestaining,ELISA,immunostaining,directorindirectPCR.Amongthosemethods,directPCRisthemostsensitive,specificandconvenientmethodwhentheprimerdesignisoptimized.
TheMycoDect™MycoplasmaDetectionKitisdesignedtodetectmycoplasmainfectionincellculturesinlessthantwohours.Itcandetectmycoplasmafrombothcelllysatesandcellculturemedia.Thesensitivityisupto10-20copiesoftargetDNA,whichtranslatestolessthan10mycoplasmaspecies.Thiskitdetectsthetopfivecommonspeciesofmycoplasma,M.Hyorhinis,M.Arginini,M.Orale,M.Fermentans,A.laidlawii,whichrepresent98%oftissuecultureinfections.ItcanalsodetectMycoplasma,Acholeplasma,UreaplasmaandSpiroplasmafromothergenera.
KitComponents
| |
ExampleofExperimentalResults | |
Sample1showsseveremycoplasmacontamination.Sample2givespositiveresultsformycoplasmawithboth270and150bpbands.Sample3givesnegativeresultswith150bpbandonly. | |
| ProductSpecifications | |
| ProductName | MycoDect™MycoplasmaDetectionKit |
| Catalog# | MD050 |
| Size | 50reactions(MD050)/200reactions(MD200) |
| Shipping | Dryiceorblueice |
| StorageandStability | Storeat-20°C.Thisproductisstablefor12monthswhenstoredasdirected. |
| QualityControl | EachlotofMycoDect™MycoplasmaDetectionKitisfunctionallytested. |
| RestrictedUse | ForResearchUseOnly.Notforuseindiagnosticortherapeuticprocedures. |
| Protocol |  Download |
- ExperimentalProcedure
- ExplanationofResults
CellsculturedintheabsenceofantibioticsformorethanthreedayscanenhancethePCRsignal.Mediasampleshouldbederivedfromcellsthatareatleast80%confluent.
SamplePreparationfromCellLysate- Aspiratemediumfromattachedcells,andrinsecellstwicewithDPBS.
- Add0.2mLoftrypsinperwellof24-wellplate,andincubateat37°Cfor2-3minutes.
- Add0.5mLof10%FBS/DMEMmedium,andtransferthecellsUSPensiontoa1.5mLmicrocentrifugetube.
- Centrifugethis1.5mLtubecontainingthecellsuspensionat300xgfor5minutesatroomtemperature.
- Aspiratethesupernatant.Washthecellpelletwith1mLDPBS.
- Centrifugethetubeagainat300xgfor5minutesatroomtemperature.
- Aspiratethesupernatant,andresuspendthecellpelletin100μLlysisbuffer.
- Lyseatroomtemperaturefor10min.
- Heatthelysatesat95°Cfor5min.
- Centrifugethelysatesat15,000xgfor5minandcollectsupernatant.
- Take1-2μLsupernatantastemplateforPCRreaction.
- Collect100µLofcellculturemediumtoamicrocentrifugetube.
- Centrifugeat15,000xgfor5min.
- Heatthecellculturemediumat95°Cfor5min.
- Take1-2µLmediumastemplateforPCRreaction.
PCRsetupwithTaqpolymeraseisshownbelow.Afinalvolumeof20μLisrecommendedforeachreaction.
| TestSample/μL | PositiveControlReaction/μL | NegativeControlReaction/μL | |
| MycoDect™PrimerMix | 15 | 15 | 15 |
| MycoDect™PCRMix | 4 | 4 | 4 |
| Sample | 1 | - | - |
| PositiveControl | - | 1 | - |
| NegativeControl | - | - | 1 |
| InTotal | 20 | 20 | 20 |
PCRProgram
| Step | Temp | Time |
| InitialDenaturation | 95°C | 4min |
| 30Cycles | 95°C | 15s |
| 56°C | 30s | |
| 68°C | 30s | |
| FinalExtension | 68°C | 5min |
| Hold | 4°C |
PCRProductDetection
Foroptimalseparationbetweenthemycoplasmabandandthecontrol,werecommend2%agarosegelforelectrophoresis.
- MixthefinalproductsofeachPCRreactionwithgelelectrophoresisloADIngbuffer.
- Loadeachsampleintoindividualwellsoftheagarosegel,includingpositivecontrol,negativecontrol,andDNAladder.
- Electrophoreseattheconditionsrecommendedbythegelboxmanufacturer.
- Visualizethebandswithethidiumbromide.
Limitations
- Useribonuclease-free(RNase-free)reagentsandsupplieswhenrunningthisassay.
- Donotmixorsubstitutereagentswiththosefromothersourcesorlots.
- Thekitshouldnotbeusedbeyondtheexpirationdateonthekitlabel.
- Anyvariationindiluents,operator,pipettingtechnique,washingtechnique,incubationtimeortemperature,andkitagecanalterassayperformance.
- Thisassaydetectsthefollowingspecies:M.hyorhinis,M.arginini,M.fermentans,M.orale,M.pirum,M.hominis,M.salivarium,andA.laidlawii.
- Anegativeresultdoesnotindicatethatotherspeciesofmycoplasmaareabsent.Additionally,mycoplasmamaybepresentatlevelsbelowthedetectablelimitsofthiskit.
- CellculturesthatarevisIBLycontaminated(i.e.,turbidityandyellowmedia)areprobablyduetoE.coliorfungalinfectionandshouldnotbetested.Visiblycontaminatedcellculturesshouldbediscardedandfreshculturesshouldbestartedfromfrozenstock.
- Thisassaycannotbeusedformycoplasmaspeciesidentification.
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