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1、3SensitiveDetectionofSARSCoronavirusRNAbyaNovelAsymmetricMultiplexNestedRT-PCRAmplificationCoupledWithOligonucleotideMicroarrayHybridizationZhi-weiZhang,Yi-mingZhou,YanZhang,YongGuo,Sheng-ceTao,ZeLi,QiongZhang,andJingChengSummaryWehavedevelopedasensitivemethodforthedetectionofspecificgene
2、ssimultaneously.First,DNAwasamplifiedbyanovelasymmetricmultiplexPCRwithuniversalprimer(s).Second,the6-carboxytetramethylrhodamine(TAMRA)-labeledPCRproductswerehybridizedspecificallywitholigonucleotidemicroarrays.Finally,matchedduplexesweredetectedbyusingalaser-inducedfluorescencescanner.T
3、heusefulnessofthismethodwasillustratedbyanalyzingsevereacuterespiratorysyndrome(SARS)coronavirusRNA.Thedetectionlimitwas100copies/μL.Theresultsoftheasymmetricmultiplexnestedreversetranscription-PCRwereinagreementwiththeresultsofthemicroarrayhybridization;nohybridizationsignalwaslostashapp
4、enedwithappliconsfromsymmetricamplifications.Thisreliablemethodcanbeusedtotheidentificationofothermicroorganisms,screeningofgeneticdiseases,andotherapplications.KeyWords:Polymerasechainreaction(PCR);multiplexPCR;asymmetricPCR;universalprimer;severeacuterespiratorysyndrome(SARS)coronavirus
5、;microarray.1.IntroductionMultiplexPolymerasechainreaction(PCR)wasdesignedtoamplifymulti-pletargetsequencesusingmorethanonepairofprimersinthereaction.Ithasthepotentialtosaveaconsiderableamountoftimeandeffortwithoutcompro-misingtestutilityandadditionalinstruments.Sinceitsfirstreportin1988(
6、1),multiplexPCRhasbecomearapidandcon-venientscreeningprocedureinbothclinicalandresearchlaboratories.Ithasbeensuccessfullyappliedtogenedeletionanalysis(2),mutationandpolymor-phismanalysis(3),mRNAquantitativeanalysis(4),RNAdetection(5,6)andFrom:MethodsinMolecularMedicine,Vol.144,Microarrays
7、inClinicalDiagnosticsEditedby:T.JoosandP.Fortina?HumanaPressInc.,Totowa,NJ5960Zhangetal.genomesequencing(7).Forinfectiousdiseasediagnosis,multiplexPCRhasbeenavaluabletoolfortheidentificationofviruses(8,9),bacteria(10,11),parasites(12),andbacterialdrug-resistancegene
