应用PCR法检测向日葵茎溃疡病菌的研究

Research on Detection of Phomopsis helianthiby PCR

【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 project aimed to designed specific primers in order to establish a rapid PCR detection methods for Phomopsis helianthi.【Method】Fungal universal primers ITS4/ITS5 were used on the PCR amplification of the bacteria.The PCR products were cloned,and went through restriction analysis and sequencing.The bacteria specific primers PHDF1 and PHDF2 were designed by DNAMAN software.And the reaction system was optimized.【Result】PCR reactions were： 25 mM MgCl2 2.4 uL,10 mM dNTP 0.3 uL,and 10 uM primers were 1.5 uL and the minimal amount of DNA was 7 ng.The best annealing temperature was 61.6℃.The rapid PCR detection method for the bacteria was established.【Conclusion】 13 strains were detected by applying this method,which showed that these primers could accurately distinguish Phomopsis helianthi bacteria from other Phomopsis bacteria,and the amplified fragment was 326 bp.