论文部分内容阅读
本研究以新疆郁金香种植区植株和进口种球为材料,利用特异性引物,对侵染郁金香的主要病毒黄瓜花叶病毒(CMV)、碎色病毒(TBV)的一步法RT-PCR分子检测技术进行了初步研究。根据两种病毒的基因序列,合成了两对特异性引物,并对RT-PCR中的引物浓度、退火温度和反应循环数等因素进行了筛选和优化。研究显示,阳性对照和带病毒植株均扩增出了与预期大小一致的特异性条带,而健康植株无任何扩增产物。一步RT-PCR的最佳反应体系为:模板RNA 1μL,2×1 Step Buffer 25μL,上、下游扩增引物各1μL,Prime Script 1 Step Enzyme Mix 2μL,加RNase Free dd H2O至50μL。黄瓜花叶病毒(CMV)和碎色病毒(TBV)特异性引物的最佳反应程序:c DNA合成50℃30 min;预变性94℃2 min,变性94℃30 s,退火(CMV)56℃/(TBV)52℃30 s,然后72℃延伸l min,30个循环,最后72℃延伸10 min。实验表明,种植区内栽培一年的郁金香植株部分已被CMV和TBV侵染,其中还有复合侵染的现象存在。本研究建立了基于核酸水平的高灵敏度的一步RT-PCR法郁金香病毒检测技术,能准确的检测出植株中的TBV和CMV,适用于郁金香黄瓜花叶病毒(CMV)、碎色病毒(TBV)的鉴定和检测,为脱毒组培苗的检测和脱毒体系的建立提供理论依据和技术支撑。
In this study, the tulip planting plant in Xinjiang and imported bulbs as materials, using specific primers, the main virus infecting tulip cucumber mosaic virus (CMV), crushing virus (TBV) by one-step RT-PCR molecular detection techniques Conducted a preliminary study. Two pairs of specific primers were synthesized according to the sequence of the two viruses, and the factors such as primer concentration, annealing temperature and reaction cycle number in RT-PCR were selected and optimized. Studies have shown that the positive control and virus-bearing plants amplified specific bands of the expected size, while healthy plants without any amplification products. The best reaction system for one-step RT-PCR was: template RNA 1μL, 2 × 1 Step Buffer 25μL, upstream and downstream amplification primers 1μL, Prime Script 1 Step Enzyme Mix 2μL, RNase Free dd H2O to 50μL. CMV and TBV specific primers: c DNA synthesis at 50 ℃ for 30 min; denaturation at 94 ℃ for 2 min, denaturation at 94 ℃ for 30 s, annealing at 56 ℃ / (TBV) at 52 ° C for 30 s followed by extension at 72 ° C for 1 min for 30 cycles and final extension at 72 ° C for 10 min. Experiments show that part of the tulip plant cultivated in the planting area has been infected by CMV and TBV, among which there are also complex infection phenomenon. This study established a highly sensitive one-step RT-PCR method for detection of tulip virus based on nucleic acid level, which can accurately detect TBV and CMV in plants, and is suitable for tulip cucumber mosaic virus (CMV), crushing virus (TBV) Identification and testing, to provide theoretical basis and technical support for the detection of virus-free tissue culture seedlings and the establishment of detoxification system.