Phytopathology. 2002 Jun;92(6):654-8. doi: 10.1094/PHYTO.2002.92.6.654.
ABSTRACT We have developed a piezoelectric DNA-sensor based on DNA-RNA hybridization for the detection of two orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). Specific oligonucleotide probes modified with a mercaptohexyl group at the 5'-phosphate end were directly immobilized onto 10-MHz AT-cut quartz crystal microbalance (QCM). QCMs coated with such oligonucleotide probes were exposed to test solutions containing viral RNA for hybridization. Various experimental conditions evaluated were (i) DNA probe coating concentration, (ii) sensitivity and specificity of the probes at different hybridization temperatures, and (iii) effects of incubation temperature on the hybridization time. The specific nucleotide probe-coated QCM-based DNA sensors were able to detect both CymMV and ORSV in quantities as low as approximately 1 ng in purified RNA preparations and 10 ng in the crude sap of infected orchids. This is the first application of a DNA biosensor for the detection of plant viruses.
摘要 我们开发了一种基于 DNA-RNA 杂交的压电 DNA 传感器，用于检测两种兰花病毒，齿兰环斑病毒（ORSV）和齿兰 mosaic 病毒（CymMV）。在 5'-磷酸末端带有巯基己基的特异性寡核苷酸探针被直接固定在 10MHz 的 AT 切石英晶体微天平（QCM）上。用这种寡核苷酸探针涂覆的 QCM 暴露于含有病毒 RNA 的测试溶液中进行杂交。评估了各种实验条件，包括（i）DNA 探针涂覆浓度，（ii）不同杂交温度下探针的灵敏度和特异性，以及（iii）孵育温度对杂交时间的影响。基于特异性核苷酸探针涂覆的 QCM 的 DNA 传感器能够在纯化 RNA 制剂中低至约 1ng 和感染兰花的粗提物中低至 10ng 的量检测到 CymMV 和 ORSV。这是 DNA 生物传感器首次用于检测植物病毒。
