一种利用水提物快速简便检测烟草中TMV的方法

摘要

Tobacco mosaic virus (TMV) is a widespread plant virus of the genus Tobamovirus that infects plants, especially tobacco and other members of the family Solanaceae. The infection causes mottling and discoloration on the leaves. In this study we reported a simple and rapid method for detection of TMV based on aqueous extract and agarose gel electrophoresis, and all procedures require 30 minutes. A small amount of tobacco leaves (～10 mg) was ground in sterile water (～100 uL), and the electrophoretic bands of about 6 000 bp in aqueous extract were detected through agarose gel electrophoresis. The Northem-blot analysis targeting TMV-replicase gene showed that the electrophoretic bands was TMV genomic. Therefore, this method is more rapid, simple and economic than those methods reported previously, and can detect the TMV of tobacco plants in 10 minutes. However, the aqueous extract is unstable under room temperature and degraded after 3 h since extraction.%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10min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3h以上,就很难再检测到特异性的电泳条带.