ExperimentalStudyonDetectionofDeadandLive Mycobacterium Tuberculosis by FlowCytometry / 中山大学学报(医学科学版)

ABSTRACT

@#：【Objective】To provide basic experimental basis for quantitative detection and analysis of antibiotic susceptibilityandheteroresistancein Mycobacterium tuberculosis byflowcytometry，weinvestigatedtheperformanceof propidiumiodide（PI）and6-carboxyfluoresceindiacetae（6-cFDA）inidentifyingdeadorlivingMycobacterium tubercu⁃ losis.【Methods】PIand6-cFDAwereusedtostain10strainsofpureculturedliving Mycobacterium tuberculosis suspen⁃ sion with turbidity of 1 McClell and the corresponding suspension with turbidity of 1 McClell heated at 85℃ for 30 minutes，respectively.Themeanfluorescenceintensity（MFI）wasmeasuredbyflowcytometry.TheMFIboundaryvalue fordistinguishingdeadandlivingMycobacterium tuberculosisweredeterminedaccordingtoreceiveroperatingcharacteristic （ROC）curves.Thesameexperimentswerecarriedoutwithother10strainsofliving Mycobacterium tuberculosis suspen⁃sionand10dead Mycobacterium tuberculosis suspension，andtheMFIthresholdwasusedtodeterminetheviabilityof bacteria.【Results】（1）IntheexperimentofdeterminingMFIboundaryvalue，theMFIof10strainsofdeadbacteria suspensionstainedwithPIwasnon-normaldistribution，themedianvaluewas2459，andtheMFIof10strainsofliving bacteriasuspensionstainedwithPIwasnormaldistribution，themeanvaluewas426±180.TheMFIboundaryvalueof deadbacteriastainedwithPIwas1329.TheMFIof10strainsofdeadbacteriaand10strainsoflivingbacteriasuspen⁃ sionstainedwith6-cFDAwasnormaldistribution，andthemeanvaluewas49±4and7144±4511，respectively.The MFIthresholdof6-cFDAstainingforlivingbacteriawas1021.（2）TheMFIthresholdsforidentifyingdeadbacteriaby PIstainingandtheMFIthresholdsforidentifyinglivingbacteriaby6-cFDAstainingwereusedtodetect10otherliving bacterialsuspensionsand10deadbacterialsuspensions.Theaccuracy，sensitivity，specificity，Yondeindex，positive predictivevalueandnegativepredictivevaluewere95%and100%，1.00and1.00，0.90and1.00，0.90and1.00，0.91 and1.00，1.00and1.00，respectively.【Conclusions】PIstainingcandetectdeadMycobacterium tuberculosisand6-cFDA stainingcandetectliving Mycobacterium tuberculosis.Identificationof Mycobacterium tuberculosis activitybasedonPIand 6-cFDAstainingwillhavebroadapplicationprospects.