10种植物病毒的基因芯片检测技术研究.pdfVIP

植物病理学报 ACTA PHYTOPATHOLOGICA SINICA 37(6)：561—565(2007) 1 0种植物病毒的基因芯片检测技术研究 马新颖 ，汪琳 ，任鲁风 ，高文娜 ，邓丛良 张永江4，吴小兵 ，相 宁4，周 琦 ( 北京出入境检验检疫局，北京100026；。本元正阳基因技术有限公司，北京100176； 。中国科学院研究生院，北京100049； 中国检验检疫科学研究院，北京100025) 摘要：本研究设计了lO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Cy3一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lO一100倍，所以，基因芯片能对植物病毒 作出快速、准确的检测。 关键词：基因芯片；植物病毒；检测 Technique for detection of 10 plant viruses by gene chip MA Xin．ying ，WANG Lin ， REN Lu—feng ，GAO Wen—na ，DENG Cong—liang ，ZHANG Yong-jiang ，wu Xiao—bing ，XIANG Ning ，ZHOU Qi ( Beijing Entry-Exit Inspection and Quarantine Bureau，Beijing 100026，China；。Benyuan Zhengyang Gene Technology Joint—Stock Company，Beijing 100176，China； Graduate of University of Chinese Academy of Sciences， Beijing 100049，China； National Academy of Inspection and Quarantine，Beijing 100025，China) Abstract： The probes of 10 plant viruses and control were designed and immobilized on chips．The total RNA was extracted by Trizol method from plants．Primers were designed according to the sequences of cp gene or rep gene of the plant viruses．The corresponding fragments were amplified by RT—PCR and the PCR products were labeled by Cy3一dCTP．Th e labeled PCR products were hybridized with the probes and the sig— nals were scanned by scanner and analyzed by GenePix Pro 4．0 software．Specific signals were identified from virus—infected plants．The detection sensitivities of gene chip were 10—100 times higher than RT—PCR．The results indicate that gene chip Can be used for detection of plant viruses fast and accurately． Key words：gene chip；plant virus；detection 中图分类号：S432．41 文献标识码：A 文章编号：0412-0914(2007)06-0561-05 基因芯片的原理是将大量DNA探针片段有 针，1次实验就可以同时分析多种生物靶点，其效 序地固化于支持物的表面，然后与已标记的生物样