Genetically Modified Herbicide-Tolerant Crops and Sugar Beet—Environmental and Health Concerns - Sweeteners: Nutritional Aspects, Applications, and Production Technology - page 377

TheqPCRisusedtodetectspeciicGMOeventsbytheusageofspeciicprimersforscreening
elementsorevent-speciicmarkers.Controlsarenecessarytoavoidfalse-positiveorfalse-negative
results.Forexample,atestforthecaulilowermosaicvirus(CaMV)isusedtoavoidafalse-positive
intheeventofavirus-contaminatedsample.
MbongoloMbellaetal.(2011)reportedSYBR ® GreenqPCRmethodsforthedetectionofendog-
enousreferencegenesincommoditycropssuchassoybean,maize,oilseedrape,rice,cotton,sugarbeet,
andpotato.EachqPCRmethodisshowntomeettheperformancecriteria(speciicity,limitofdetection,
andPCReficiency)setbytheEuropeanNetworkofGMOLaboratories(ENGL).Whencombinedwith
theequivalentqPCRmethodstargetingGMOelements,thesecrop-speciicSYBRGreenqPCRmethods
canaidthedevelopmentofaneficienttoolfordeterminingGMOpresenceinfoodandfeedproducts.
An overview of the different techniques applied is presented in the work of Querci et al. (2010).
Manyoftheseapproachesusetwosteps:(1)screeningforthepresenceofcropsandGMmaterialusing
common genetic elements present in authorized GMOs (CaMV 35S promoter and tNOS) and (2) a
GMO identiication step where event-speciic methods are used to identify which GMOs are present
(Marmirolietal.2008).
Leimanis et al. (2008) used a microarray platform for a combination of crop, trait-, GM ele-
ment-,construct-,andevent-speciicGMOscreeningtargets.Morissetetal.(2008)usedtheNASBA
implementedmicroarrayanalysis(NAIMA),anucleicacidsequence-basedampliicationapproach
adaptedonanarrayplatformtodevelopamultitargetGMOscreeningtool.
Real-time quantiication of the GMOs involves the choice of adequate primers, probes, and
evaluation model, the appropriate standard material, and the DNA isolation method. The DNA
extraction method should be cost and time effective, especially in the case of high amounts of
samples.Previousexaminationsofdifferentcommerciallyavailablekitsbasedonsilicagel,mag-
neticbeads,andprecipitationwerecomparedtolysisandprecipitationwithCTAB,showingthatthe
conventionalCTABpuriicationgavethehighestyieldwithsuficientPCRampliicationprotocols
(Holdenetal.2003).Besidesthereal-timeexperiments,Kunertetal.(2006)havealsosequenced
the 162-bp-long amplicon of the CaMV 35S promoter and found 100% homology for BT11,
NK603,andMON810,givingevidenceforthequantiicationofrealsampleswiththeseprimers.
Additionally,theycomparedtheampliconsoftheinvertaseexonfromthreeoftheirbreedingswith
theGenbankentry(GI1122438).InthecasesofNK603andMON810,theyfoundsomesequence
divergenceleadingtodifferentPCReficienciesthatresultedinproblemswiththeinterpretationof
quantiication(ArvanitoyannisandVarzakas2006).
Panetal.(2006)developedanevent-speciicdetectionmethodbasedonthelankingsequenceof
anexogenousintegrantinthetransgenicmaizeMON863thatcontainsthecry3Bb1geneexpressinga
Bacillus thuringiensis Cry3Bb1 protein that is selectively toxic to a maize root worm pathogen. The
3ʹ-integration junction between the host plant DNA and the integrated DNA of transgenic MON863
maizewasisolatedusingthermalasymmetricinterlaced-PCR.Theevent-speciicprimersandTaqMan
probeweredesignedbasedontheisolated30-integrationjunctionsequence,andqualitativeandquan-
titativePCRsystemsemployingthesedesignedprimersandprobewereestablished.Inthissystem,the
limitofdetectionofthequalitativePCRassaywasestimatedtobe40initialhaploidcopies.Thelimit
ofquantitationofthequantitativePCRassayinauthenticMON863maizeseedswasestimatedtobe
approximately80haploidcopies.GMMON863contentswerealsoquantiiedrelativetotheendogenous
maizestarchsynthaseIIb(zSSIIb)geneDNA,andtheresultswereexpressedasthepercentageofthe
GMMON863maizeDNArelativetothetotalcontentofthemaizeDNA.
QualitativeandquantitativeanalyticalmethodsweredevelopedfortheneweventofGMmaize
MON863byLeeetal.(2006).
Biotechnologicaladvanceshavepavedtheirwayforveryeffectiveandreliableauthenticitytest-
ing.Thelattercanbefocusedeitheronvarietyandgeographicalorigindeterminationortraceability
testingofawiderangeoffoodproducts—producesofagriculturalandanimaloriginandpackage
counterfeiting.ApartfromthewidelyemployedDNAmethods,otherinstrumentalmethodsinclude