Anovelmethodbasedonhighresolutionmelting(HRM)analysisforMIRU–VNTRgenotypingofMycobacteriumtuberculosisYuPanga,YangZhoua,ShengfenWanga,JieLub,BingLua,GuangxueHea,LixiaWanga,⁎,YanlinZhaoa,⁎aNationalCenterforTuberculosisControlandPrevention,ChineseCenterforDiseaseControlandPrevention,Beijing,ChinabInstituteofAnimalandPlantQuarantine,ChineseAcademyofInspectionandQuarantine,Beijing,ChinaabstractarticleinfoArticlehistory:Received14February2011Receivedinrevisedform16May2011Accepted26May2011Availableonline13June2011Keywords:MycobacteriumtuberculosisMIRU–VNTRGenotypingHRMThemycobacterialinterspersedrepetitiveunit–variablenumbertandemrepeat(MIRU–VNTR)methodisoneofthemostimportantmethodsthathavebeenusedinrecentyearsforgenotypingMycobacteriumtuberculosis.Agarosegelelectrophoresisandcapillaryelectrophoresishavebeenusedtodeterminethesizeofamplicons,however,bothofthesemethodshaveshortcomings.Here,wedevelopandevaluateanovelmethodforMIRU–VNTRtypingbasedonhighresolutionmelting(HRM)analysis.TheMIRU40locuswasselectedtoevaluatedifferentreal-timePCRmachinesandtheaccuracyofourmethod;theRocheLightCycler480providedgreatestconsistencybetweentheTmvalueandrepeatnumberandwasusedinsubsequentevaluations.Ourmethodgivesgreateraccuracyincomparisonwithconventionalagarosegelelectrophoresis(98.9%vs.90.9%,p=0.017),and,withthehelpoffittingformulae,canbeusedtoobtainthenumberofMIRUtandemrepeatsfromtheTmvalue.Tovalidateourmethodweanalyzed12classicalMIRUlocitogenotype88clinicalisolates.ThenumberofMIRUtandemrepeatswasdeterminedaccurately,quicklyandconveniently.CrownCopyright©2011PublishedbyElsevierB.V.Allrightsreserved.1.IntroductionGenotypingofMycobacteriumtuberculosis(MTB)hasbeenusedextensivelytounderstandthetransmissionandprevalenceofdifferentMTBstrains(Djelouadjietal.,2008;Mostrometal.,2002;VanSoolingen,2001),andhasprovidedsignificantknowledgecontributingtotubercu-losiscontrolandprevention.Inlinewithrecentadvancesinmoleculartechniques,variousgenotypingmethodshavebeendevelopedtodifferentiateclin...
