Venor®GeMqEPutilizesquantitative,real-timePCR(qPCR).Thekitcanbeperformedwithanytypeofreal-timePCRcyclerabletodetectthefluorescencedyesFAM™andHEX™.Theprotocolprovidedispreferredforfastandreliablescreeningofcellculturesupernatantsmostapplicableinresearchanddevelopment.Thedetectionprocedurecanbeperformedwithin3hours.

MycoplasmasarespecificallydetectedbyamplifyingahighlyconservedrRNAoperon,ormorespecifically,the16SrRNAcodingregioninthemycoplasmagenome.Themycoplasma-specificamplificationisdetectedat520nm(FAM™channel).ThekitincludesprimerandFAM™labeledprobeswhichallowthespecificdetectionofallMollicutesspeciessofardescribedascontaminantsofcellculturesandmediacomponents.EukaryoticDNAisnotamplifiedbythisprimer/probesystem.
FalsenegativeresultsduetoPCRinhibitorsorimproperDNAextractionaredetectedbytheinternalamplificationcontrolwhichcanbeaddedtothePCRmastermix.Theamplificationoftheinternalamplificationcontrolisdetectedat610nm(HEX™channel).

TypeofPCR

TaqMan®based qPCRAssaywithFAM™andHEX™labeledprobes

RecommendedUse/Scope

Venor®GeMqEPisusedfordirectdetectionofMollicutes(Mycoplasma,Acholeplasma,Spiroplasma)contaminationincellculturesandcellmediacomponents.

KitComponents

lyophilizedprimer/nucleotide/probe/polymerase/mixinaliquotsof25tests
rehydrationbuffer

lyophilizedinternalamplificationcontrol
lyophilizedpositivecontrol
PCRgradewater

PackageSizes

• Cat.No.11-902525tests
  Cat.No.11-9100100tests
  Cat.No.11-9250250tests

ResultEvaluation

cyclerbased,real-timePCR

RequiredConsumables

PCRreactiontubes
OptionalforprocessvalidationandEP2.6.7complianttesting:
InternalControlDNAextra(4vialsfor300µleachofinternalamplificationcontrol;Cat.No.11-9905)
10CFU™SensitivityStandardsavailableforallEP 2.6.7listedmycoplasmaspecies

RequiredLabDevices

qPCRcyclerwithFAM™andHEX™filter
variablemicroliterPipettes
benchtopcentrifugefor1.5mlreactiontubes

ShelfLifeandStorage

Componentscanbestoredat+2to+8°Cforatleast12months.Afterrehydrationthereagentsmustbestoredat-18°C.

EP2.6.7Compliance

Yes.

Pleasenotethatvalidationdataareprovidedforinformationpurposeonly.EP2.6.7clearlystates“Wherecommercialkitsareused…,documentedvalidationpointsalreadycoveredbythekitmanufacturercanreplacevalidationbytheuser.Nevertheless,theperformanceofthekitwithrespecttoitsintendedusehastobedemonstratedbytheuser(e.g.detectionlimit,robustness,cross-detectionofotherclassesofbacteria.”.Pleasefeelfreetocontactusifyouneedfurtherassistance.

Fig.AmplifieddilutionseriesofMycoplasmafermentans,performedona
CorbettRotorGene®6000.