Highly Sensitive Fluorescent-labeled Probes and Glass Slide Hybridization for the Detectio

Highly Sensitive Fluorescent-labeled Probes and Glass Slide Hybridization for the Detection of Plant

RNA Viruses and a Viroid

Zhiyou DU;Bo JIN;Wenhong LIU;Liang CHEN;Jishuang CHEN

【期刊名称】《生物化学与生物物理学报：英文版》 【年(卷),期】2007(039)005

【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 5×saline sodium citrate (SSC) as a spotting solution. Using a CY5-labeled 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