霉菌 毒素 基因芯片

JOURNALOFCLINICALMICROBIOLOGY,0095-1137/00/$04.0010Feb.2000,p.781–788Vol.38,No.2Copyright©2000,AmericanSocietyforMicrobiology.AllRightsReserved.RapidDiagnosisofBacteremiabyUniversalAmplificationof23SRibosomalDNAFollowedbyHybridizationtoanOligonucleotideArrayR.M.ANTHONY,T.J.BROWN,ANDG.L.FRENCH*DepartmentofMicrobiology,King’sCollegeSt.Thomas’Campus,St.Thomas’Hospital,LondonSE17EH,UnitedKingdomReceived10May1999/Returnedformodification8September1999/Accepted29October1999Therapididentificationofbacteriainbloodculturesandotherclinicalspecimensisimportantforpatientmanagementandantimicrobialtherapy.Wedescribearapid(4h)detectionandidentificationsystemthatusesuniversalPCRprimerstoamplifyavariableregionofbacterial23SribosomalDNA,followedbyreversehybridizationoftheproductstoapanelofoligonucleotides.Thisprocedurewassuccessfulindiscriminatingarangeofbacteriainpurecultures.Whenthisprocedurewasapplieddirectlyto158unselectedpositivebloodculturebrothsonthedaywhengrowthwasdetected,125(79.7%)werecorrectlyidentified,including4withmixedcultures.Nine(7.2%)yieldedbacteriaforwhichnooligonucleotidetargetswerepresentintheoligo-nucleotidepanel,and16culture-positivebroths(10.3%)producednoPCRproduct.Insevenoftheremainingeightbroths,streptococciwereidentifiedbutnotsubsequentlygrown,andoneisolateofStaphylococcusaureuswasmisidentifiedasacoagulase-negativestaphylococcus.Theaccuracy,range,anddiscriminatorypoweroftheassaycanbecontinuallyextendedbyaddingfurtheroligonucleotidestothepanelwithoutsignificantlyincreasingcomplexityorcost.Theisolationofbacteriafrombloodcultures(bacteremia)isusuallyindicativeofaseriousinvasiveinfectionrequiringur-gentantimicrobialtherapy.Differentorganismshavedifferentantimicrobialsusceptibilities,andsuccessfultreatmentisde-pendentonthepromptadministrationofthecorrectdrug(6,14,23,25,26,33).Bloodculturebrothsusuallybecomeposi-tive8to24hafterinoculation.Atthistime,someindicationofbacterialidentitycanbeobtainedbyGramstaining,butdefin-itiveidentificationandantibioticsusceptibilitiesareusuallynotavailableuntil24to48hlater.Thisdelayhastwoconse-quences:first,thepatientmaysufferifineffectivetherapyisgivenforantibiotic-resistantorganisms,andsecond,antibioticresistancemaybeencouragedifunnecessaryantibioticsaregivenforsensitiveorganisms(3,21).Althoughinourhospitalninebacterialgroups(coagulase-negativestaphylococci[CoNS],Escherichiacoli,Staphylococcusaureus,Pseudomonasaerugi-nosa,Enterococcusspp.,Klebsiellaspp.,Enterobacterspp.,Pro-teusspp.,andStreptococcuspneumoniae)accountformorethanhalfofallclinicallysignificantbloodcultureisolates,asmanyas50speciesmaybeinvolved,andthereareusuallyfewclinicalcluesastothespecificcausativeorganism.Rapidspe-ciesdetectionandidentificationwouldfacilitateearliereffec-tivetherapy.Rapiddiagnosiscanbeachievedbythedirectdetectionofcharacteristicbacterialgenesinclinicalspecimens,andmanyprimersetshavebeendevelopedtodetectspecies-specificgenesinsimplePCRs(10,18,31).ThesesystemsareusuallydesignedtoconfirmthediagnosisofspecificclinicalsyndromesandincludetheidentificationofBurkholderiapseudomalleiinmelioidosis(9,34),S.pneumoniaeinpneumococcalpneumo-niaandmeningitis(7,22),CoxiellaburnettiiinQfever(46),Listeriamonocytogenesinlisteriosis(5),Rhodococcusequiinrhodococcosisinhorses(42),Mycobacteriumtuberculosisintuberculosis(12,36),SalmonellaentericaserovarTyphiinty-phoidfever(38,44),Mycoplasmapneumoniaeinmycoplasmalpneumonia(30),Neisseriameningitidisinmeningococcalmen-ingitis(31),andBorreliaburgdorferiinLymedisease(15).Sim-ilartestsystemshavebeendesignedforyeasts(29).Inmostcases,thePCRhasbeenperformedonpositivebloodculturebottlesamples,butsomehavebeensuccessfulwithdirectbloodsamples,serum,buffycoatspecimens,ornegativebloodculturebottlesamples;theseresultssuggestthatDNA-basedmethodsmaybemoresensitivethanconventionalbacteriologymethods.However,theuseofdifferentprimersfordifferentspeciesisimpracticalfortheroutineanalysisofbloodculturesthatmaycontainoneormoreofmanypossiblepathogens.EitheracomplexPCRwithamixtureoflargenumbersofprimersisneeded,oralargeseriesofindividualPCRsmustberuninparallelorsequentially.MultiplexPCRmaybeeffectiveforalimitednumberoforganisms,butasmoreprimersareadded,thesensitivitydecreasesandthechancethattwounrelatedprimerswillproducespuriousproductsincreases.Multiplein-dividualPCRsincreasetheexpenseandcomplexityoftheassayand,iftheyarerunsequentially,theprocessingtimeincreasesforlesscommonorunexpectedpathogens.TheseproblemscanbeavoidedbyusingasinglepairofuniversalprimersdesignedtoamplifyconservedstretchesofDNAfromanybacteriumpresent,followedbysequenceanal-ysisofthePCRproducttodeterminethespecies.Previousinvestigatorshaveusuallychosenthe16SribosomalDNA(rDNA)orthe16S-23SrDNAspacerregionasatargetforuniversalprimers(35).The16SrDNAishighlyconserved,andsequencesfromitarenowusedinbacterialtaxonomy(45).Incontrast,the16S-23SrDNAspacerregionishighlyvariablewithinmanyspecies,frequentlycontainingtRNAgenes,andthisvariationhasbeenusedfortypingclinicalisolates(2,18,41).Therehavebeenafewreportsoftheuseof16SrDNAvariationforthedetection