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甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)是引起我国玉米矮化叶病的主要病毒。根据其基因组序列,SCMV可以分为4个组,其中第IV组分离物属于新出现的强毒株系。为了快速检测SCMV尤其是IV组分离物的发生情况,我们针对SCMV I-IV组及IV组分离物设计了6对引物,从中筛选出特异性最强的2对引物(I-IV-2F/I-IV-2R和IV-1F/IV-1R),进行PCR体系的优化。优化后的PCR体系为:退火温度51℃,d NTP终浓度为0.1 m M,引物终浓度为0.20μM,Taq DNA聚合酶终浓度为0.015 U/μL。利用该体系,引物对I-IV-2F/I-IV-2R和IV-1F/IV-1R均能够从50 ng感病叶片或0.05 ng病毒RNA中检测出SCMV。通过优化两对引物浓度比例建立了能同时检测SCMV所有分离物及第四组分离物的双重PCR体系。利用该体系从山东、河南及云南均检测出SCMV第IV组分离物的发生。本研究为SCMV尤其是SCMV第IV组分离物的快速检测提供了技术支持。
Sugarcane mosaic virus (SCMV) is the major virus causing maize dwarf leaf disease in China. According to their genomic sequences, SCMV can be divided into four groups, of which the group IV isolates belong to the newly emerged virulent strains. In order to rapidly detect the occurrence of SCMV, especially IV group isolates, we designed six pairs of primers for SCMV I-IV and IV isolates and selected the two pairs of primers with the highest specificity (I-IV-2F / I-IV-2R and IV-1F / IV-1R) were used to optimize the PCR system. The optimized PCR system consisted of an annealing temperature of 51 ° C, a final dNTP concentration of 0.1 mM, a final primer concentration of 0.20 μM, and a final Taq DNA polymerase concentration of 0.015 U / μL. Using this system, primer pairs I-IV-2F / I-IV-2R and IV-1F / IV-1R were able to detect SCMV from 50 ng of infected leaves or 0.05 ng of viral RNA. By optimizing the ratio of two primer pairs, a dual PCR system was established to detect all SCMV isolates and the fourth group of isolates at the same time. The system was used to detect the occurrence of SCMV IV isolates from Shandong, Henan and Yunnan. This study provided technical support for the rapid detection of SCMV, especially SCMV Group IV isolates.