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【目的】设计出向日葵茎溃疡病菌(Phomopsis helianthi M.Muntanola-Cvetkovic)的特异性引物,建立该病菌PCR快速检测方法。【方法】用真菌通用引物ITS4/ITS5对向日葵茎溃疡病菌进行PCR扩增,将扩增产物进行克隆、酶切分析和测序,用DNAMAN软件设计出检测该病菌的特异性引物PHDF1和PHDF2,优化PCR反应体系。【结果】PCR反应体系为:25 mM MgCl22.4μL,10 mM dNTP 0.3μL,10 uM引物各1.5μL,模板7ng,最佳退火温度61.6℃,建立了该病菌PCR检测方法。【结论】应用该方法检测供试的13个菌株,该对引物可以准确的将向日葵茎溃疡病菌与其他拟茎点霉属的真菌区分开来,该病菌的PCR扩增片段为326 bp。
【Objective】 The objective of this study was to design a specific primer for Phomopsis helianthi M.Muntanola-Cvetkovic and establish a rapid PCR detection method for this bacterium. 【Method】 The fungal common primers ITS4 / ITS5 were used to amplify the sunflower stem canker. The amplified products were cloned, digested and sequenced. The DNA primers were designed to detect the specific primers PHDF1 and PHDF2, and optimized PCR reaction system. 【Result】 The PCR reaction system consisted of 22.4 μL of 25 mM MgCl 2, 0.3 μL of 10 mM dNTP, 1.5 μL of 10 μM primer, 7ng template and the optimal annealing temperature of 61.6 ℃. The PCR assay was established. 【Conclusion】 This method was used to detect 13 strains tested. The primers can distinguish the sunflower stem canker bacteria from other fungi of Phomopsis spp. Accurately. The PCR amplified fragment of this strain is 326 bp.