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1、大豆根腐病致病镰砲菌的多重PCR检测技术何宛芹付瑶鲁雯璐常小丽杨文锤四川农业大学农学院四川省作物带状复合种植工程技术研究中心摘要：为建立大豆根腐病镰他菌的多重PCR检测方法，以四川大豆根腐病致病菌包括尖也镰也菌、腐皮镰孑包菌、禾谷镰刀菌和木贼镰泡菌为对象，设计镰泡菌翻译延伸因子基因EF-la的种特异引物，建立多重PCR扩增体系，并进行优化与验证。结果表明:25uL体系为最优镰孑包菌多重PCR扩増体系，4种镰抱菌等体积混合DNA4.0nL,各镰抱菌特异正向引物l.OuL,共用反向引物4.0nL,最佳退火温度为54°C,当循环30次时，能清
2、晰地扩增出各镰抱菌EF-la条带，对4种镰也菌混合DNA的检测灵敏度可达0.1ng/卩Lo室内环境样木验证结果表明，依据EF-la扩增片段大小，该体系能够特异地检测出大豆黄化苗与致病镰砲菌混合样本屮的镰抱菌，但无法从其它真菌的DNA屮扩增获得目的片段。表明基于EF-la基因特异引物建立的镰砲菌多重PCR检测技术可快速、特异地检测大豆根腐病镰他菌。关键词：人豆根腐病;镰孑包菌;多重PCR;翻译延伸因子基因;病原菌鉴定;作者简介：常小丽，E-mail:xl_cha.ngkit@126.com;作者简介：杨文乍玉，E-mai1:mssiyan
3、gwy@sicau.edu.cn收稿日期：2016-12-26基金：四川省科技支撑项目(2015NZ0040)AmultiplexPCRdetectiontechniqueforthepathogenicFusariumspeciescausingsoybeanrootrotHeWanqinFuYaoLuWenluChangXiaoliYangWenyuSichuanEngineeringResearchCenterforCropStripIntercroppingSystem,CollegeofAgriculture,SichuanA
4、griculturalUniversity;Abstract：ToestablishamultiplexPCRdetectiontechniqueforFusariumspeciescausingsoybeanrootrot,thefourmajorpathogenicFusariumspecies,Fusariumoxysporum,F.solani,F・equisetiandF・graminearum,insouthwestChinawereselected.Basedonthesequeneeofthetranscriptione
5、longationfactorgeneEFTci,theFusariumspecies-specificprimersweredcsigned,andthemultiplexPCRwereestablished.Thereactionandamplificationparameterswereoptimized,andthesensitivityandspecificitywereverified.TheresultsshowedthatallspecificEFTagenefragmentsoffourFusariumspeciesw
6、ereamplifiedbythemultiplexPCRinatotalvolumeof25uLwith4.0uLmixedDNA,1.0uLspecificforwardprimerforeachFusariumspecies,and4.0uLcommonreverseprimeratanannealingtemperatureof54°Cand30cycles.ThemultiplexPCRsystemwascapableofdetectingthemixedDNAwithaconcentration0.1ng/uL.Thepat
7、hogenicFusariumspecieswassuccessfullytestedfromthemixedsamplesofsoybeanetioldtedseed!ingswithFusariumspeciesbyusingtheoptimizedmultipicPCRaccordingtofragmentsizeofEFTagene,whereasthisgenewasnotamplifiedfromDNAsamplesofothertestedfungi.ThestudyindicatedthatthemultiplexPCR
8、techniquebasedonspecificEFTageneprimerpairsoffourspeciesofFusariumcouldbeusedfortherapidandprecisedetec
