Highly sensitive fluorescent-labeled probes and glass slide hybridization for the detection of plant RNA viruses and a viroid.
In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing (32)P 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-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 (32)P-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.
Du
Zhiyou
Z
College of Life Sciences, Zhejiang University, Hangzhou 310029, China.
Jin
Bo
B
Liu
Wenhong
W
Chen
Liang
L
Chen
Jishuang
J
D002951:Citrates / Q000494:pharmacology; D015342:DNA Probes / Q000737:chemistry*; D005189:False Positive Reactions; D005456:Fluorescent Dyes / Q000494:pharmacology*; D005898:Glass; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009757:Nylons / Q000737:chemistry; D010942:Plant Viruses / Q000737:chemistry*; D010944:Plants / Q000821:virology*; D012313:RNA / Q000737:chemistry; D012328:RNA Viruses / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D012821:Silanes / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D000077559:Sodium Citrate; D014772:Viroids / Q000737:chemistry
D002951:Citrates; D015342:DNA Probes; D005456:Fluorescent Dyes; D009757:Nylons; D012367:RNA, Viral; D012821:Silanes; D000077559:Sodium Citrate; D012965:Sodium Chloride; D012313:RNA