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本研究旨在建立1种有效而灵敏的方法来同时检测粮谷中玉米褪绿斑驳病毒、小麦线条花叶病毒、玉米矮花叶病毒、高原小麦花叶病毒。选取WSMV L2/R2、MDMV 482F/R、MCMV 384F/R、HPWMoV283F/R分别作为检测小麦线条花叶病毒、玉米矮花叶病毒、玉米褪绿斑驳病毒、高原小麦花叶病毒的引物。当退火温度为59℃，延伸温度为66℃，反应体系中玉米褪绿斑驳病毒、小麦线条花叶病毒、玉米矮花叶病毒、高原小麦花叶病毒引物终浓度分别为0.2μmol/L、0.2μmol/L、0.2μmol/L、0.32μmol/L时，达到最好扩增效果。以4种混合阳性病毒质粒为模板开展灵敏度检测，发现其检测限可达1 fg/μL。选取103个测试样品进行检测，检出1种及以上病毒的样品有42个，实现单反应管有效、准确检测4种检疫性病毒，为田间、口岸实验室快速检测鉴定检疫性病毒提供便利。
Abstract：The aim of this research was to establish an effective and sensitive method for the simultaneous detection of maize chlorotic mottle virus(MCMV),wheat streak mosaic virus(WSMV),maize dwarf mosaic virus(MDMV) and High Plains wheat mosaic virus(HPWMoV) in grains. Primer pairs of WSMV L2/R2,MDMV 482F/R,MCMV 384F/R,and HPWMoV 283F/R were selected for the detection of wheat streak mosaic virus,maize dwarf mosaic virus,maize chlorotic mottle virus and High Plains wheat mosaic virus,respectively. The optimal amplification effect was achieved when the annealing temperature was 59℃,the extension temperature was 66℃,and the final concentrations of maize chlorotic mottle virus,wheat streak mosaic virus,maize dwarf mosaic virus and High Plains wheat mosaic virus primers in the reaction system were 0.2 μmol/L,0.2 μmol/L,0.2 μmol/L,0.32 μmol/L,respectively. The sensitivity test was carried out using the plasmids of the four mixed-positive viruses as templates,and the detection limit was found to be as high as 1 fg/μL. A total of 103 samples were selected for the test,and 42 samples tested positive for one or more viruses,realizing the effective and accurate detection of the four quarantine viruses in a single reaction tube,which is convenient for the rapid detection and identification of quarantine viruses in the field and the port laboratories.
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基本信息:
DOI：10.19662/j.cnki.issn1005-2755.2025.03.002
中图分类号:S41-30
引用信息:
[1]张海鹏,黄艳,于翠等.利用多重RT-PCR检测进境粮谷中的4种病毒[J].植物检疫,2025,39(03):6-14.DOI:10.19662/j.cnki.issn1005-2755.2025.03.002.
基金信息:
广东省口岸安全智能化检测重点实验室资助（2023B1212010011）