A Simple and Rapid DNA Purification Method for Detection of Leishma- nia DNA in Peripheral Blood of Patients with Visceral Leishmaniasis.

[摘要] Introduction:RecentlypolymerasechainreactiontechniqueisbeingusedfordetectionofleishmaniaDNAinclinicalsamplesofpatientssuspectedwithvisceralleishmaniasis.Sincemanyofthepatientsarechildrenandalsosufferingfromanemiaandleucopenia,thereisaneedforthedevelopmentofaDNAextractionmethodusingsmallamountofclinicalsamples.Inthepresentstudy,asimpleandrapidmethodwasintroducedforperformingtheDNAextractionprocedurewhichwascapableofremovinginhibitorsfromthewholebloodsam-plesandofprovidingtheappropriateDNAforLeishmaniaPCRamplification.MaterialsandMethods:EDTA-peripheralbloodspecimenswerecollectedfromhealthyvolunteersandfrozenat-20°C.ThethawedspecimenswerespikedwithadefinitenumberofL.infantumpromastigotes.Usinginhousemadelysisbufferandboiling,aDNAextractionmethodwasdevelopedforthepurificationofLeishmaniagenomefrom500IJlofbloodsamples.Thedevelopedmethodwasthencomparedwiththeclassicalstandardmethodtoobtainthehighestsensitivityforthediagnosisofvisceralleishmaniasis.Results:ThesensitivityofthePCRforthedevelopedmethodwas10copiesofL.infantumDNAperreactiontube.Whenclinicalsamplescollectedfrompatientswithadefinitediagnosisofvisceralleishmaniasiswereused,nofalsenegativewasdemonstratedwithnewlydevelopedmethod.Conclusion:Sincehazardousmaterialssuchasphenol-chloroformarenotusedduringextractionprocedure,itseemstobemuchsaferthanthestandardphenol-chloroformmethod.However,theuseofsmallamountofsample(i.e.0.5IJl)andremovalofPCR-inhibitorsfromthewholebloodarethemajoradvantagesofourdevel-opedDNAextractionmethod.

[学科分类] 基础医学

[关键词] Leishmania;DNA purification;Peripheral blood.