Rapid Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis Using a Multiplex Real-Time PCR Assay 英文参考文献.docVIP

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Rapid Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis Using a Multiplex Real-Time PCR Assay 英文参考文献.doc

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Rapid Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis Using a Multiplex Real-Time PCR Assay 英文参考文献

RapidDifferentialDiagnosisbetweenExtrapulmonary TuberculosisandFocalComplicationsofBrucellosis UsingaMultiplexReal-TimePCRAssay Mar?′aIsabelQueipo-Ortun?o1,2*,JuanD.Colmenero3,PilarBermudez4,Mar?′aJose′ Bravo5,PilarMorata1,2 1Biochemistry and Molecular Biology Department, Faculty of Medicine, University of Malaga, Malaga, Spain, 2CIBER Fisiopatolog?′a Obesidad y Nutricio′n (CB06/03) InstitutodeSaludCarlosIII,Madrid,Spain,3InfectiousDiseasesService,CarlosHayaUniversityHospital,Malaga,Spain,4MicrobiologyService,CarlosHayaUniversity Hospital,Malaga,Spain,5ImmunologyService,CarlosHayaUniversityHospital,Malaga,Spain Abstract Background: Arduous to differ clinically, extrapulmonary tuberculosis and focal complications of brucellosis remain importantcausesofmorbidityandmortalityinmanycountries.Wedevelopedandappliedamultiplexreal-timePCRassay (MRT-PCR)forthesimultaneousdetectionofMycobacteriumtuberculosiscomplexandBrucellaspp. Methodology:ConventionalmicrobiologicaltechniquesandMRT-PCRforM.tuberculosiscomplexandBrucellasppwere performedon45clinicalspecimensfrompatientswithfocalcomplicationsofbrucellosis orextrapulmonarytuberculosis and 26 control samples. Fragments of 207bp and 164bp from the conserved region of the genes coding for an immunogenicmembraneproteinof31kDaofB.abortus(BCSP31)andtheintergenicregionSenX3-RegX3wereusedfor theidentificationofBrucellaandM.tuberculosiscomplex,respectively. Conclusions:ThedetectionlimitoftheMRT-PCRwas2genomesperreactionforbothpathogensandtheintra-andinter- assay coefficients of variation were 0.44% and 0.93% for Brucella and 0.58% and 1.12% for Mycobacterium. M RT-PCR correctlyidentified42ofthe45samplesfrompatientswithtuberculosisorbrucellosisandwasnegativeinallthecontrols. Thus,theoverallsensitivity, specificity, PPVandNPV valuesof theMRT PCR assaywere93.3%, 100%,100%and89.7%, respectively,withanaccuracyof95.8%(95%CI,91.1%–100%).SinceMRT-PCRishighlyreproducibleandmorerapidand sensitive than conventional microbiological