葡萄霜霉病菌环介导等温扩增检测体系的建立

Development of LAMP detection system for Plasmopara viticola

建立了葡萄霜霉病菌(Plasmopara viticola)环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测体系,为葡萄霜霉病的早期诊断和预测预报提供依据。根据葡萄霜霉病菌的rDNA-ITS全长基因序列设计合成了7对引物,建立并优化了葡萄霜霉病菌LAMP反应体系。利用该检测体系对包括P.viticola在内的14种常见植物病原真菌的基因组进行LAMP扩增,验证所建立体系的特异性,并对灵敏性进行评价。使用优化后的检测体系对田间发病叶片的总DNA基因组进行扩增,确认了所建立的检测体系的实用性。通过对比7对引物的特异性,筛选出1对特异性良好的引物。LAMP最佳反应条件为:以PVITFIP-1/PVITBIP-1和PVITF3-1/PVITB3-1为引物,反应体系中Bst DNA聚合酶含量为0.32 U·μL-1,在62℃反应60 min可以检出葡萄霜霉病菌DNA,其灵敏度为0.3 ng·μL-1。初步在田间使用该技术,可以检测到葡萄霜霉病菌,说明所建立的体系具有一定的实用性。构建的葡萄霜霉病菌LAMP检测体系高效,特异性强,可以用于检测田间的葡萄霜霉病菌。

A LAMP(loop-mediated isothermal amplification)detection system of Plasmopara viticola was established to provide a scientific basis for early diagnosis and prediction of grape downy mildew.According to the rDNA-ITS gene sequence of P.viticola in GenBank,seven pairs of specific primers were designed to establish and optimize the LAMP detection system.LAMP amplification was performed on the genome of 14 kinds of plant pathogens including P.viticola to verify the specificity of the established system and evaluate the sensitivity.The practicability of the established system was verified by amplifying the genome of diseased leaves in the field by optimized detection system.A pair of primers with good specificity was selected by comparing the specificity of 7 pairs of primers.The optimum conditions for LAMP of P.viticola were obtained.Taking PVITFIP-1/PVITBIP-1 and PVITF3-1/PVITB3-1 as primers,the concentration of Bst DNA polymerase was 0.32 U·μL-1.The DNA of P.viticola could be detected at the reaction temperature of 62℃for 60 min,and the detected sensitive was at the level of 0.3 ng·μL-1.Preliminary application of this technology in the field could detect P.viticola,indicating the established system has certain practicality.This study demonstrated that the LAMP system was not only high effective but also strong specificity for the detection of P.viticola,which could be apply to detect the P.viticola in the field.