Selection and Application of ssDNA Aptamers to Detect Active TB from Sputum Samples 英文参考文献.docVIP

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Selection and Application of ssDNA Aptamers to Detect Active TB from Sputum Samples 英文参考文献.doc

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Selection and Application of ssDNA Aptamers to Detect Active TB from Sputum Samples 英文参考文献

SelectionandApplicationofssDNAAptamerstoDetect ActiveTBfromSputumSamples LiaS.Rotherham1,2,CharlotteMaserumule1,3,KeertanDheda3,JacquesTheron2,MakobetsaKhati1,3* 1EmergingHealthTechnologiesPlatform,CouncilforScientificandIndustrialResearch,BiosciencesUnit,Pretoria,SouthAfrica,2DepartmentofMicrobiologyandPlant Pathology,UniversityofPretoria,Pretoria,SouthAfrica,3DepartmentofMedicine,GrooteSchuurHospitalandUniversityofCapeTown,CapeTown,SouthAfrica Abstract Background:Despitetheenormousglobalburdenoftuberculosis(TB),conventionalapproachestodiagnosiscontinueto relyonteststhathavemajordrawbacks.TheimprovementofTBdiagnosticsrelies,notonlyongoodbiomarkers,butalso uponaccuratedetectionmethodologies.The10-kDaculturefiltrateprotein(CFP-10)andthe6-kDaearlysecretedantigen target(ESAT-6)arepotentT-cellantigensthatarerecognisedbyover70%ofTBpatients.Aptamers,anovelsensitiveand specificclassofdetectionmolecules,hashitherto,notbeenraisedtotheserelativelyTB-specificantigens. Methods:DNAaptamersthatbindtotheCFP-10.ESAT-6heterodimerwereisolated.Toassesstheiraffinityandspecificityto the heterodimer, aptamers were screened using an enzyme-linked oligonucleotide assay (ELONA). One suitable aptamer wasevaluatedbyELONAusingsputumsamplesobtainedfrom20TBpatientsand48controlpatients(thosewithlatentTB infection,symptomaticnonTBpatients,andhealthylaboratoryvolunteers).CulturepositivityforMycobacteriumtuberculosis (Mtb)servedasthereferencestandard.Accuracyandcut-pointswereevaluatedusingROCcurveanalysis. Results: Twenty-four out of the 66 aptamers that were isolated bound significantly (p,0.05) to the CFP-10.ESAT-6 heterodimer and six were further evaluated. Their dissociation constant (KD) values were in the nanomolar range. One aptamer,designatedCSIR2.11,wasevaluatedusingsputumsamples.CSIR2.11hadsensitivityandspecificityof100%and 68.75%usingYouden’sindexand35%and95%,respectively,usingarule-incut-point. Conclusion: This preliminary proof-of-concept study suggests that a diagnosis of active TB u