一种快速、特异性检测小麦中链格孢菌（Alternaria alternata）的巢式 PCR 方法
일충쾌속、특이성검측소맥중련격포균（Alternaria alternata）적소식 PCR 방법
A Rapid and Specific Nest-PCR Method for Detection of Alternaria alternata in Wheat
  • 万方数据
    河南农业科学 하남농업과학
    JOURNAL OF HENAN AGRICULTURAL SCIENCES
    2014年 10期 67-73 ,共7页

    秦召%李巧云%段宗彪%姜玉梅%牛吉山

    진소%리교운%단종표%강옥매%우길산

    小麦%链格孢菌%检测%巢式 PCR 小麥%鏈格孢菌%檢測%巢式 PCR
    소맥%련격포균%검측%소식 PCR
    wheat%Alternaria alternata%detection%nest-PCR
    为建立检测小麦中链格孢菌（Alternaria alternata ）的方法，从河南省小麦黑胚籽粒上分离得到7个优势 A．alternata 病原菌株，对其 rDNA 的内部转录间隔区序列（ITS）进行测序，序列比较表明，分离到的病原菌株与 GenBank 公布的 A．alternata 的 ITS 区序列一致性达到99％～100％。根据 Alternaria 属菌株（A．alternata、A．tenuis、A．tenuissima）和麦类根腐离蠕孢菌（Bipolaris sorokiniana ）的 ITS 序列比对结果，设计出1对 A．alternata 特异性引物 Aa1F/Aa1R。利用多种河南省小麦的主要病原真菌对该引物的特异性进行验证，只有在 A．alternata 中能特异性地扩增出1条443 bp 的 DNA 片段。以真菌通用引物 ITS1/ITS4为外侧引物、特异性引物Aa1F/Aa1R 为内侧引物进行巢式 PCR 扩增，能检测到 A．alternata DNA 的最低质量浓度为50 pg/μL。因此，建立的巢式 PCR 方法能够快速、特异地检测小麦中的 A．alternata。
    위건립검측소맥중련격포균（Alternaria alternata ）적방법，종하남성소맥흑배자립상분리득도7개우세 A．alternata 병원균주，대기 rDNA 적내부전록간격구서렬（ITS）진행측서，서렬비교표명，분리도적병원균주여 GenBank 공포적 A．alternata 적 ITS 구서렬일치성체도99％～100％。근거 Alternaria 속균주（A．alternata、A．tenuis、A．tenuissima）화맥류근부리연포균（Bipolaris sorokiniana ）적 ITS 서렬비대결과，설계출1대 A．alternata 특이성인물 Aa1F/Aa1R。이용다충하남성소맥적주요병원진균대해인물적특이성진행험증，지유재 A．alternata 중능특이성지확증출1조443 bp 적 DNA 편단。이진균통용인물 ITS1/ITS4위외측인물、특이성인물Aa1F/Aa1R 위내측인물진행소식 PCR 확증，능검측도 A．alternata DNA 적최저질량농도위50 pg/μL。인차，건립적소식 PCR 방법능구쾌속、특이지검측소맥중적 A．alternata。
    The aim of this study was to establish a method for detection of Alternaria alternata in wheat.Seven dominating isolates of A.alternata from wheat black point grains in Henan province were collected,and then their internal transcribed spacer(ITS)regions in rDNA were sequenced, and the sequence analysis showed that the ITS sequences were highly similar among isolates of A.alternata isolated in this study and in GenBank by 99%-100%.According to the comparative result of ITS sequences of Alternaria spp.(A.alternate,A.tenuis,A.tenuissima)and Bipolaris sorokiniana,a pair of primers,namely Aa1F and Aa1R,were designed.Specificity of the primers was tested with several major fungal pathogens of wheat in Henan province,and a 443 bp specific DNA fragment could be amplified only from A.alternata.A rapid and specific nest-PCR method for detection of A.alternata in wheat was established with fungal universal primers ITS1/ITS4 as the outer primers and Aa1F/Aa1R as the inner primers.The nest-PCR method was used to detect the samples with gradated concentrations of A.alternata DNA,and the minimum DNA concentration detected was 50 pg/μL.
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