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由板栗疫病菌(Cyphonectria parasitica)引起的板栗疫病是板栗栽培区的主要病害,也是全国林业危险性有害生物。为建立C.parasitica的快速分子检测技术,根据C.parasitica与Gen Bank中同属其他物种的ITS序列差异设计了引物CP1/CP2,扩增了大小为285 bp的目的片段。进一步用RAPD技术从供试菌株中标记出C.parasitica的特异性片段,通过对RAPD特异片段克隆、测序后设计引物SC1/SC2,扩增的目的片段大小为389bp,实现了RAPD标记向SCAR标记的成功转化。采用引物对组合方式将引物CP1/CP2和SC1/SC2组成双重PCR,优化PCR反应条件并检测引物的特异性和灵敏度。双重PCR能从C.parasitica扩增出285 bp和389 bp的两条特异条带,而其他供试菌株及阴性对照均无条带,检测灵敏度达到300 fg/μL的基因组DNA。利用双重PCR能成功检测出自然条件下已发病板栗及处于潜伏期病害中的C.parasitica。
Chestnut blight caused by Cyphonectria parasitica is a major disease in the chestnut cultivation area and also a national forestry pest. In order to establish the rapid molecular detection technique of C. parasitica, CP1 / CP2 primers were designed according to the ITS sequence differences between C. parasitica and Gen Bank in other species. A 285 bp fragment was amplified. The specific fragment of C.parasitica was further identified by RAPD from the tested strains. The specific fragment of RAPD was cloned and sequenced to design a primer SC1 / SC2. The size of the amplified fragment was 389bp. The successful conversion. The primers CP1 / CP2 and SC1 / SC2 were double-stranded PCR to optimize the PCR reaction conditions and detect the specificity and sensitivity of the primers. Double PCR amplified two specific bands of 285 bp and 389 bp from C. parasitica, while other test strains and negative control showed no band, and the detection sensitivity was 300 fg / μL genomic DNA. The use of dual PCR can successfully detect C. chinensis under natural conditions and C. parasitica in the incubation period.