Medical Microbiology

216 3 GeneralBacteriology

MolecularMethods........................................

Themainobjectiveofthemolecularmethodsofbacterialidentificationisdi-

rectrecognitionofpathogen-specificnucleotidesequencesinthetestmate-

rial.Thesemethodsareusedinparticularinthesearchforbacteriathatare

notculturable,areverydifficulttoculture,orproliferateveryslowly.Of

course,theycanalsobeusedtoidentifypurebacterialcultures(seeabove).

Inprinciple,anyspecies-specificsequencecanbeusedforidentification,but

thespecificregionsofgenescodingfor 1 6SrRNAand23SrRNAareparticu-

larlyusefulinthisrespect.Thefollowingmethodsareused:

&DNAprobes.SinceDNAismadeupoftwocomplementarystrandsofnu-

cleicacids,itispossibletodetectsingle-strandsequenceswiththehybridi-

zationtechniqueusingcomplementarymarkingofsinglestrands.Theprobes

canbemarkedwithradioactivity(^32 P,^35 S)ornonradioactivereportermole-

cules(biotin,dioxigenin):

— Solidphasehybridization.Thereportermoleculeorprobeisfixedtoany-

lonornitrocellulosemembrane(colonyblottechnique,dotblottechni-

que).

— Liquidphasehybridization.Thereportermoleculeandprobeareinasolute

state.

— In-situhybridization.DetectionofbacterialDNAininfectedtissue.

&Amplification.Themainobjectivehereistoincreasethesensitivitylevel

soastofindthe“needleinahaystack.”Anumberoftechniqueshavebeen

developedtodate,whichcanbeclassifiedinthreegroups:

— Amplificationofthetargetsequence.Theoldestandmostimportantamong

thetechniquesinthisgroupisthepolymerasechainreaction(PCR),which

isdescribedonp.409f.).With“realtimePCR,”avariantofPCR,theanal-

ysiscanbecompletedin 10 minutes.

— Probeamplification.

— Signalamplification.

IdentificationbyMeansofAmplificationandSequencing

Thetargetsequenceforidentificationofbacteriathathavenotyetbeencultured

(e.g.,Tropherymawhipplei,thecausalpathogeninWhipple’sdisease)orofpatho-

gensverydifficulttoidentifywithclassicmethods,isoftenacertainregionofthe

1 6SrRNA,somesectionsofwhichareidenticalinallbacteria.Betweenthesehighly

conservativesegmentsareothersectionsthatarespecificforaspeciesorgenus.

Usingprimersthatcanrecognizetheconservedregionsof 1 6SrDNAtotheright

andleftofthespecificregions,thespecificsequenceisamplified,thensequenced.

Thebasesequencethusobtainedisthenidentifiedbycomparisonwithareference

datalibrary.

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Kayser, Medical Microbiology © 2005 Thieme