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1、单条线虫DNA提取方法王江岭1张建成2顾建锋1收稿日期：基金项目：国家质量监督检验检疫总局科技项目（2010IK269）；宁波市农业科研攻关合作项目(2008C10014)；宁波市自然科学基金项目（2010A610018）作者简介：王江岭（1982—），男，河南开封人，助理农艺师，硕士，主要从事检疫性植物病原线虫的检测鉴定.E-mail:jiangling0916@foxmail.com通讯作者：顾建锋（1972-），男，浙江余姚人，高级农艺师，大学本科，从事检疫性植物病原线虫及真菌的检测鉴定.Tel：0574-8702
2、2839；E-mail:gujf@nbciq.gov.cn.（1宁波出入境检验检疫局技术中心宁波3150122嘉兴出入境检验检疫局嘉兴314000）OptimizedmMethodofextractDNAfromasinglenematode.WangJiangling1,ZhangJiancheng2,GuJianfeng1*（1NingboEntry-exitInspectionandQuarantineBureau,Ningbo315012;2JiaxingEntry-exitInspectionandQuaran
3、tineBureau,Jiaxing314000）AbstractTherearemanyproblemsinextractingDNAfromlargenumberofnematodes.Thenematodescouldbeamixedspecie.TheextractedDNAweredecreasinginexperimentprocess.Amplificationmightbecumberedbyaddingmoresolutes,andeventoeffectonthenextexperiments.Int
4、hismethodasinglenematodewasfreezedbyliquidnitrogenandheatedat85℃todisruptethestructureofnematodecellsbychangingthetemperaturesuddenly.ThentheproteinsweredissolvedeasilybyproteinaseKandmoreDNAwereextracted.,Thismethodwasprovedeffective,rapid,andstabilebykindsofnem
5、atodes.thePCRresultisgoodevenwhentheextractedDNAweredilutedfor32time.ComparedwithtwootherproteinaseKmethods,toextractnematodeDNA,thismethodwasprovedeffective,rapid,andstabile,andcouldbeusedinrapidtest.NematodeproteinsweredissolvedquitelyandreleasedmoreDNA.Morecha
6、ncestodomolecularexperimentswereprovided.Theadvantagesofthismethodare:(1)PCRBufferattachedtoTaqenzymetaketheplaceofWLBandSDSlysisliquids,whichreducestheinstabilityofreactionsystemandtheeffectofaddedsolutes.(2)AlltheprocessisfinishedinonePCRtube,noliquidtransporte
7、dandlesssolutesadded.SothepollutionandbadeffecttothelaterPCRprocessaregreatlyreduced.(3)Manualdisruptionofnematodeisreplacedbypoikilothermictreatment,;andtheeffectofpollutionisavoidedandnoexperimenttechniqueisrequired.ComparedwithtwootherproteinaseKmethodsofnemat
8、odeDNAextraction,thismethodwasprovedeffectiveandstabile,andcouldbeusedinrapidtest;nematodeproteinsweredissolvedcompletelyandmoreDNAreleased,thePCRresultisgoode
