甘蔗花叶病毒第Ⅳ组分离物RT-PCR检测体系的建立及应用

Optimization and application of RT-PCR detection systemfor Sugarcane mosaic virus group Ⅳ isolates

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 mM,引物终浓度为0.20μM,Taq DNA聚合酶终浓度为0.015 U·μL^(-1)。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 prevalent virus inducing maize dwarf mosaic disease in China. According to their complete genome sequences, SCMV isolates are divided into four groups, among which group IV is highly virulent. In order to rapidly detect the SCMV isolates, especially those of group IV, we de- signed 6 pairs of primers specific for SCMV groups I-IV and group IV, respectively. Primer pairs I-IV-2F/I-IV- 2R and IV-1F/IV-1R which showed the highest specificity were selected for optimization of RT-PCR detection system. The results of optimization were annealing temperature 51℃, the final concentrations of dNTP, primers and Taq DNA polymerase were 0.1 mM, 0.2 μM and 0.015 U · μL^-1, respectively. With the optimized system and primer pairs I-IV-2F/I-IV-2R and IV-IF/IV-I, one can detect SCMV from 50 ng diseased leaves and 0.05 ng viral RNA. A duplex PCR system was established by adjusting the concentration of two pairs of primers. U- sing this detect system, isolates of SCMV group IV were detected from Shandong, Henan and Yunnan provinces. This study provides technical support for the rapid detection of SCMV especially the fourth group.