水稻黑条矮缩病毒RT-LAMP快速检测方法的建立

doi:10.3864/j.issn.0578-1752.2012.07.006

摘要/Abstract

摘要： 【目的】建立一种快速、灵敏的逆转录环介导等温扩增方法（RT-LAMP）检测寄主植物和传毒介体体内的水稻黑条矮缩病毒（Rice black-streaked dwarf virus，RBSDV）。【方法】合成4条针对RBSDV S10核苷酸序列6个位点的特异性引物。分别对引物浓度、MgSO4浓度、反应温度和时间进行优化。将感病水稻总RNA梯度稀释后进行灵敏性检验并与RT-PCR比较分析。选择RBSDV和南方水稻黑条矮缩病毒（SRBSDV）验证该方法的特异性。用本RT-LAMP方法检测田间病株。【结果】RT-LAMP检测方法可排除SRBSDV的干扰而特异地检测植物和飞虱体内的RBSDV，与RT-PCR灵敏性基本一致。检测结果易于判定。【结论】RT-LAMP检测方法适合寄主植物和介体体内RBSDV的快速检测。

关键词: 水稻黑条矮缩病毒, RT-LAMP, 检测

Abstract: 【Objective】The objective of this study is to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of Rice black-streaked dwarf virus (RBSDV) from host plants and insect vector.【Method】Four primers matching a total of 6 sequences of the S10 of RBSDV were synthesized for the RT-LAMP assay. The concentration of the primer and MgSO4 were optimized, and the best temperature and reaction time for detecting the virus were found, respectively. Detection sensitivity comparisons were performed between the RT-LAMP and RT-PCR assay using a single extraction of total RNA from RBSDV-infected rice leaves which was serially diluted in ten-fold increments in DEPC-treated water. In order to determine the specificity of the RT-LAMP assay, total RNA of rice leaves infected with RBSDV or Southern rice black-streaked dwarf virus (SRBSDV) was applied separately to the reaction system. RBSDV-infected rice plants collected from rice field were detected.【Result】 This method demonstrated a high degree of specificity for RBSDV, which can distinguish RBSDV from SRBSDV. The method was also proved to be extremely sensitive, which was as much as the RT-PCR for RBSDV detection. The detection of amplified products was easily monitored.【Conclusion】The RT-LAMP assay is suitable for rapid detection of RBSDV in host plant and vectors.

Key words: Rice black-streaked dwarf virus, RT-LAMP, detection

周彤, 杜琳琳, 范永坚, 周益军. 水稻黑条矮缩病毒RT-LAMP快速检测方法的建立[J]. 中国农业科学, 2012, 45(7): 1285-1292.

ZHOU Tong, DU Lin-Lin, FAN Yong-Jian, ZHOU Yi-Jun. Development of a RT-LAMP Assay for Rapid Detection of Rice black-streaked dwarf virus[J]. Scientia Agricultura Sinica, 2012, 45(7): 1285-1292.

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