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本研究设计了10种植物病毒和各种对照的探针,将探针固定在醛基化玻璃片基上制备基因芯片。用常规的Trizol法提取植物总RNA,根据病毒的外壳蛋白基因或复制酶基因设计特异性引物,经RT-PCR扩增相应区段并用Cy3-dCTP进行标记。将标记的PCR产物与芯片杂交,扫描仪对杂交结果扫描,GenePix Pro 4.0软件对杂交图像进行分析。结果表明,该芯片可以从病毒感染样本中检测到特异性识别信号,检测灵敏度比RT-PCR高10~100倍,所以,基因芯片能对植物病毒作出快速、准确的检测。
In this study, 10 kinds of plant viruses and various control probes were designed and probes were immobilized on aldehyde-based glass substrates to prepare gene chips. The plant total RNA was extracted by the conventional Trizol method. Specific primers were designed according to the coat protein gene or replicase gene of the virus. The corresponding segments were amplified by RT-PCR and labeled with Cy3-dCTP. Hybridization of the labeled PCR product with the chip, scanning of the hybridization results by the scanner, and analysis of hybridization images by GenePix Pro 4.0 software. The results showed that the chip can detect specific recognition signals from virus-infected samples and the detection sensitivity is 10 to 100 times higher than that of RT-PCR. Therefore, the gene chip can rapidly and accurately detect plant viruses.