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3种大白菜病毒多重RT-PCR检测技术的建立
刘 欢,李春游,刘 斐,等
作者单位
刘 欢 西北农林科技大学 旱区作物逆境生物学国家重点实验室农业部西北黄土高原作物有害生物综合治理重点实验室植保资源与病虫害治理教育部重点实验室 
李春游 西北农林科技大学 旱区作物逆境生物学国家重点实验室农业部西北黄土高原作物有害生物综合治理重点实验室植保资源与病虫害治理教育部重点实验室;周至县农业技术推广中心 
刘 斐,等 咸阳市农业科学研究院 
摘要:
【目的】建立3种大白菜病毒病多重RT-PCR检测技术,为病毒病快速检测、白菜病毒病防治与抗病育种提供参考。【方法】针对我国大白菜芜菁花叶病毒(Turnip mosaic virus,TuMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)3种病毒的外壳蛋白(CP)基因保守区序列,设计特异性检测引物,从多重RT-PCR(mRT PCR)扩增引物、Mg2+浓度、Taq DNA聚合酶及dNTPs浓度、退火温度、延伸时间和循环次数等方面对RT-PCR检测技术进行优化。【结果】在dNTPs浓度为0.24 mmol/L,Mg2+浓度为2.8 mmol/L,Taq DNA聚合酶浓度为0.12 U/μL,退火温度为52 ℃,延伸时间为30 s,循环30次,即可成功地在一个体系中对TuMV、TMV和CMV 3种病毒复合侵染的大白菜病样进行多重RT-PCR扩增,扩增得到228,305和460 bp 3条特异性条带,其大小与试验设计相符合。灵敏度接近单重RT-PCR(sRT-PCR),体现了多重RT-PCR的优越性。【结论】建立了能同时检测大白菜样品中TuMV、TMV和CMV的多重RT-PCR检测体系,该多重RT-PCR方法能对田间大白菜样品进行3种病毒的准确、快速、高效检测。
关键词:  大白菜  芜菁花叶病毒  烟草花叶病毒  黄瓜花叶病毒  多重RT-PCR检测
DOI:
分类号:
基金项目:公益性行业(农业)专项“蔬菜主要病毒病防控技术研究与示范”(201303028);高等学校学科创新引智计划项目(B07049)
Simultaneous detection of three viruses infecting Chinese cabbage by multiplex RT-PCR
LIU Huan,LI Chunyou,LIU Fei,et al
Abstract:
【Objective】Chinese cabbage virus diseases commonly occurred in China,causing serious yield losses.In order to quickly detection of these viruses and making foundation to disease control and breeding for disease resistance to guide the disease resistant breeding.【Method】A multiplex reverse transcription polymerase chain reaction (mRT PCR) system was established for simultaneous detection of Turnip mosaic virus (TuMV),Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) in Chinese cabbage.Three sets of specific primers were designed according to the coat protein (CP) genes of the three viruses,respectively.The concentrations of the primers,Mg2+,Taq DNA polymerase and dNTPs were tested and the PCR conditions including annealing temperatures and amplification cycles were optimized.【Result】Expected fragments of 228 bp (TuMV),305 bp (TMV) and 460 bp (CMV) were amplified by this mRT-PCR system which dNTPs concentration was 0.24 mmol/L,Mg2+ concentration was 2.8 mmol/L,Taq DNA polymerase concentration 0.12 U/μL,annealing temperature was 52 ℃,extension of time was 30 s and 30 cycles.With similar detection sensitivity to each single RT-PCR (sRT-PCR).【Conclusion】The mRT PCR detection system for TuMV,TMV and CMV was successfully established.This system can accurately and quickly determine the infection of the three viruses from Chinese cabbage samples.
Key words:  Chinese cabbage  Turnip mosaic virus  Tobacco mosaic virus  Cucumber mosaic virus  detection of multiplex RT PCR