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1、干血滤纸片试剂盒和水煮法提取间日疟原虫DNA用于PCR检测的比较蚌埠医学院2011年8月第36卷第8期[文章编号]1000-2200(2011)08-0793-03干血滤纸片试剂盒和水煮法提取间日疟原虫DNA用于PCR检测的比较793?基础医学?胡明洁,吴守伟,刘辉,张静,方强.,沈继龙[摘要]目的:比较水煮法和干血滤纸片基因组DNA分离试剂盒提取间日疟原虫DNA用于PCR检测及克隆研究的差异.方法:采集间日疟患者末梢血制备干血滤纸片,分别用水煮法和QIAampDNAminikit试剂盒提取间日疟原虫基因组DNA10份.PCR扩增LDH基因
2、,并克隆质粒pGEM—PvLDH.分析比较2种提取方法的差异.结果:水煮法和试剂盒法提取的间日疟原虫gDNA,均扩增出LDH基因特异条带,但试剂盒提取的gDNA目的条带亮于水煮法.结论:水煮法操作简便,快速,经济,在等量血源条件下,所得DNA量较少,纯度较低;试剂盒提取可获得较高的得率,在定量检测和复合扩增时受到的影响因素较少,成功率较高.[关键词]间日疟原虫;DNA提取;干血滤纸片;聚合酶链式反应[中国图书资料分类法分类号]R382.31[文献标识码]AAcomparativestudyofdriedbloodspotgenomicDNA
3、isolationkitandboilingmethodinDNAextractionandPCRdetectionofPlasmodiumvivaxHUMing-jie,WUShou—wei,LIUHui,ZHANGJing,FANGQiang,SHENJi—long(.DepartmentofMicrobiology,AahuiMedicalUniversity,HefeiAnhui230031;2.DepartmentofBioscience,3.DepartmentofMicrobiology,BengbuMedicalColleg
4、e,BengbuAnhui,233030,China)[Abstract]0bjecfive:Tocomparethedifferencesofdriedbloodspot(DBS)genomicDNAisolationkitandtheboilingmethodinDNAextraction,PCRandcloningofPlasmodiumvivax.Methods:Theperipheralbloodwascollectedfromvivaxmal耐apatientsandthedriedbloodspotswereprepared.
5、ThegenomicDNAoftensampleswasextractedwithboilingmethodandQIAampDNAminikit(QIAGEN,Germany).Thelactatedehydrogenase(LDH)genewasamplifiedbyusingtheextractedDNAastemplate.TheamplifiedfragmentwaspurifiedandclonedintopGEMvector.Thedifferencesofbothmethodswereanalyzedandcompared.
6、Results:ThetargetfragmentofLDHgenewasamplifiedwiththegDNAextractedbythebothmethods;buttheelectrophoreticbandswiththetemplatebykitmethodwereobviouslybrighterthanthatbyboilingmethod.Conclusions:Theboilingmethodisasimple,rapidandeconomicalwayinextractinggDNAfromthedriedbloods
7、pots;butinagivenamountofbloodsample,theextractionrateandpurityofDNAarelower;whiletheextractionkit,withahigherextractionrate,islessaffectedbyinthequantitativeanalysisormuhiplexPCR.[Keywords]Plasmodiumvivax;DNAextraction;driedbloodspots;polymerasechainreaction疟疾是一种严重危害人类健康的感
8、染性疾病,据WHO2010年发布的数据显示,全球受到间日疟原虫感染威胁的人口高达26亿,我国大部分流行区均以间日疟流行为主.因为不会导致类似于恶性疟的高病死率,问日疟常常被认为是