Highly Sensitive Fluorescent-labeled Probes and Glass Slide Hybridization for the Detection of Plant RNA Viruses and a Viroid  

作　　者：Zhiyou DU Bo JIN Wenhong LIU Liang CHEN Jishuang CHEN 

机构地区：[1]College of Life Sciences, Zhejiang University, Hangzhou 310029, China [2]Institute of Bioengineering, Zhejiang Sci-Tech University, Hangzhou 310018, China [3]College of Life Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China [4]College of Bioengineering, Zhefiang Chinese Medical University, Hangzhou 310053, China

出　　处：《Acta Biochimica et Biophysica Sinica》2007年第5期326-334,共9页生物化学与生物物理学报（英文版）

摘　　要：In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing 32p labels with CY5 labels and replacing nylon membranes with positive-charged glass slides, for detecting plant RNA viruses and a viroid. The modified RNA dot-blot hybridization method was named glass slide hybridization. The optimum efficiency of RNA binding onto the surfaces of activated glass slide was achieved using aminosilane-coated glass slide as a solid matrix and 5xsaline sodium citrate （SSC） as a spotting solution. Using a CY5-1abeled DNA probe prepared through PCR amplification, the optimized glass slide hybridization could detect as little as 1.71 pg of tobacco mosaic virus （TMV） RNA. The sensitivity of the modified method was four times that of dot-blot hybridization on nylon membrane with a 32P-labeled probe. The absence of false positive within the genus Potyvirus [potato virus A, potato virus Y （PVY） and zucchini yellow mosaic virus] showed that this method was highly specific. Furthermore, potato spindle tuber viroid （PSTVd） was also detected specifically. A test of 40 field potato samples showed that this method was equivalent to the conventional dot-blot hybridization for detecting PVY and PSTVd. To our knowledge, this is the first report of using dot-blot hybridization on glass slides with fluorescent-labeled probes for detecting plant RNA viruses and a viroid.In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing 32p labels with CY5 labels and replacing nylon membranes with positive-charged glass slides, for detecting plant RNA viruses and a viroid. The modified RNA dot-blot hybridization method was named glass slide hybridization. The optimum efficiency of RNA binding onto the surfaces of activated glass slide was achieved using aminosilane-coated glass slide as a solid matrix and 5xsaline sodium citrate （SSC） as a spotting solution. Using a CY5-1abeled DNA probe prepared through PCR amplification, the optimized glass slide hybridization could detect as little as 1.71 pg of tobacco mosaic virus （TMV） RNA. The sensitivity of the modified method was four times that of dot-blot hybridization on nylon membrane with a 32P-labeled probe. The absence of false positive within the genus Potyvirus [potato virus A, potato virus Y （PVY） and zucchini yellow mosaic virus] showed that this method was highly specific. Furthermore, potato spindle tuber viroid （PSTVd） was also detected specifically. A test of 40 field potato samples showed that this method was equivalent to the conventional dot-blot hybridization for detecting PVY and PSTVd. To our knowledge, this is the first report of using dot-blot hybridization on glass slides with fluorescent-labeled probes for detecting plant RNA viruses and a viroid.

关 键 词：glass slide hybridization fluorescent labeling dot-blot hybridization VIRUS VIROID 

分 类 号：S432.41[农业科学—植物病理学]