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由烟草普通花叶病毒(Tobacco mosaic virus,TMV)侵染所致的植物病毒病是世界性的难题,具有寄主广泛,易于传播等特点。笔者研究了一种快速简捷检测烟草中TMV的方法,通过利用无菌水研磨TMV侵染后发病的烟草病叶,取上清液进行1%的琼脂糖凝胶电泳检测,可检测到一条特异性的,大小为6 000 bp的电泳条带,而在健康烟草叶片水提物中却检测不到该电泳条带的存在。以TMV复制酶基因片段为探针,对该特异性电泳条带进行Northern-blotting分析。结果表明,水提物中的电泳条带与TMV复制酶基因探针发生较强的杂交信号,暗示该条带可能为TMV的基因组。因此,该方法可在10 min内方便快速的检测烟株中TMV的存在,避免了常规血清学检测和分子检测(RT-PCR和western-blot)等方法的昂贵试剂和繁琐的操作步骤。但是该方法的水提取物在室温下不稳定,当静止3 h以上,就很难再检测到特异性的电泳条带。
Plant virus disease caused by Tobacco mosaic virus (TMV) infection is a worldwide problem. It has the characteristics of extensive host and easy dissemination. A rapid and simple method for the detection of TMV in tobacco has been studied. By using sterile water to mulch the diseased tobacco leaves infected with TMV, the supernatant was subjected to 1% agarose gel electrophoresis to detect a specific The size of the electrophoresis band was 6 000 bp, while the presence of the electrophoresis band was undetectable in the aqueous extract of healthy tobacco leaves. Using TMV replicase gene fragment as a probe, the specific electrophoresis bands were analyzed by Northern-blotting. The results showed that the electrophoresis band in water extract and TMV replicase gene probe strong hybridization signal, suggesting that the band may be TMV genome. Therefore, this method can detect the presence of TMV in tobacco plants easily and quickly within 10 min, avoiding the expensive reagents and cumbersome procedures of routine serological and molecular detection (RT-PCR and western-blot). However, the water extract of this method is not stable at room temperature. When it is still for more than 3 h, it is difficult to detect the specific electrophoresis band.