【摘要】 为建立牛传染性鼻气管炎病毒(IBRV)和赤羽病病毒(AKAV)的双重快速检测方法,本研究根据IBRV gB基因和AKAV S基因序列设计合成了2对特异引物和探针,建立了同时检测这两种病毒的基因芯片检测方法,即以合成探针的5’端添加poly T10尾并氨基化固定于经处理的玻璃基片表面作为捕捉序列,采用荧光素Cy3标记正向引物的5’端,对样品的PCR扩增产物进行检测,并通过试验确定其适宜杂交温度为38℃。该方法检测IBRV和AKAV重组质粒的灵敏度分别为3.6×103拷贝/25μL和9.26×104拷贝/25μL,同时通过对牛的腺病毒、牛病毒性腹泻病毒、牛水泡性口炎病毒、口蹄疫病毒及牛白血病病毒等的检测均无交叉反应,表明其特异性强。该检测方法的建立对于加强进出口牛的IBRV和AKAV检验检疫具有十分重要的意义。更多还原

【Abstract】 To establish a rapid duplex assay for detection of infectious bovine rhinotracheitis virus(IBRV) and Akabane virus(AKAV), based on two pairs of primers(labeled with Cy3 at 5’ end of forward primers) and probes(added 10 polyT with animation modification at 5’ end) designated according to IBRV gB S gene and AKAV S gene sequences in GenBank, the gene chip assay was developed by spotting the probe oligonucleotides on the glass support as capture sequences to specifically detect the PCR products for the 2 kinds of virus. Under the optimized of hybridization condition of 38 ℃, the detection limits of the gene chip was 3.6×103copies/25 μL for IBRV and 9.26×104copies/25 μL for AKAV, respectively, and no cross-reaction with other bovine virus. The chip was proved to be useful for laboratory rapid detections and import-export quarantines of IBRV and AKAV infected in cattle.更多还原