摘要
【目的】建立南瓜病毒病双重RT-PCR检测方法,检测南瓜多种病毒病.【方法】根据GenBank登录的WMV和ZYMV等病毒外壳蛋白基因序列,设计两对特异性引物,以复合感染4种病毒的南瓜病叶组织为材料,进行双重RT-PCR检测体系的优化.【结果】调查发现武威美洲南瓜病毒病种类有西瓜花叶病毒(WMV)、小西葫芦黄花叶病毒(ZYMV)、南瓜花叶病毒(SqMV)、烟草花叶病毒(TMV),症状类型有黄化、蕨叶、疱斑、黄化、疱斑混合症状、蕨叶、疱斑混合症状、蕨叶、疱斑、黄化混合症状.双重RT-PCR共检测到两种病毒ZYMV和WMV,双重RTPCR的最佳优化体系为:dNTPs浓度为0.3mmol/L,Taq DNA聚合酶浓度为0.5U,Mg^(2+)浓度为1.0mmol/L,最佳退火温度为50℃.双重RT-PCR检测体系验证表明从复合感染WMV和ZYMV的南瓜病叶上同时能够检测到这两种病毒,且片段大小分别为485和280bp的外壳蛋白.【结论】初步建立和优化了美洲南瓜病毒病双重RTPCR检测体系.
【Objective】To establish duplex RT-PCR methods to detect multiple virus in Cucurbita pepo.【Method】Specific primers were designed according to the genome sequences of WMV and ZYMV which were published in Gen Bank.The leaves of C.pepoinfected by 4virus species were selected as the experimental materials,it is used as template for duplex RT-PCR.【Result】The survey found multiple virus existed in C.pepofrom Wuwei,including watermelon mosaic virus(WMV),small zucchini yellow mosaic virus(ZYMV)and squash mosaic virus(SqMV),tobacco mosaic virus(TMV).The symptom types showed yellow,fern leaf,blister spot,yellowing,blister spot symptoms,fern leaf,blister spot mix symptoms,fern leaf,blister spot,yellowing mixed symptoms.The duplex RT-PCR detected two kinds of virus,ZYMV and WMV.The detection condition of duplex RT-PCR were optimized as dNTPs at 0.3mmol/L,Taq of DNA polymerase at 0.5U,Mg2+ at 1.0mmol/L and TM at 50℃.Duplex RT-PCR system showed that the sequence of 485 and 280bp were detected simultaneously from the composite infection WMV andZYMV’s C.pepo infected leaves.【Conclusion】The duplex RT-PCR system for C.pepo virus was established and optimized.
作者
郭银潇
郑勇民
徐秉良
刘佳
张文军
张树武
薛应钰
沈志彦
GUO Yin-xiao;ZHENG Yong-min;XU Bing-liang;LIU Jia;ZHANG Wen-jun;ZHANG Shu-wu;XUE Ying-yu;SHEN Zhi-yan(College of Plant Protection,Gansu Agricultural University,Lanzhou 730070,China)
出处
《甘肃农业大学学报》
CAS
CSCD
北大核心
2018年第6期150-156,共7页
Journal of Gansu Agricultural University
基金
国家自然科学基金项目(31460518)
省科技厅支撑计划(1104NKCA074)
甘肃农业大学盛彤笙科技创新基金项目(GSAU-STS-1420)
