猪蓝耳
发布时间：2018-07-20 11:07
【摘要】：猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome, PRRS)、猪瘟(Classical swine fever, CSF)、流行性乙型脑炎(Japanese encephalitis, JE)是三种常见的引起猪繁殖障碍疾病,在生产猪场存在混合感染的情况较多。因此,同步快速鉴别诊断这三种病具有重要意义。基因芯片技术适合高通量鉴别诊断,但传统的基因芯片标记技术多数会用到荧光素,且需要价格昂贵设备,不适合基层单位的广泛应用。本研究开展了PRRSV-CSFV-JEV直观可视化基因芯片的构建,并进行了临床应用评价,为猪繁殖障碍病的诊断提供了新的基因芯片技术。1. PRRSV-CSFV-JEV可视化共检基因芯片的构建研究：选取PRRSV的Y11基因、CSFV的PS8基因和JEV的C基因设计探针,λDNA作为阳性质控基因,探针和质控基因用乙醇醋酸钠法纯化,稀释到400ng/uL,以点样液等体积稀释到200ng/uL,用喷样的方式将探针基因和阳性质控基因按照设计阵列喷点到氨基基片上,并重复2次。点制完成的基片在预设为80℃的烘箱中烘焙4h,紫外交联30min,密封、4℃保存。PRRSV-CSFV-JEV可视化共检基因芯片的杂交流程包括靶基因的扩增标记、预杂交、杂交、Nanogold-Streptavidin孵育和银染显色判定结果。对可视化基因芯片的杂交温度、杂交时间、Nanogold-Streptavidin的稀释倍数、银染显色的时间进行探索和优化,并对该芯片的特异性、灵敏性、稳定性和保存期进行验证。结果表明PRRSV-CSFV-JEV可视化共检基因芯片在48℃杂交60min,Nanogold-Streptavidin稀释40倍,银染时间8-10min效果最好。PRRSV-CSFV-JEV可视化共检基因芯片特异性良好,探针之间无交叉反应,灵敏度可达16.2pg/uL,重复性良好,4℃密封至少可保存180d。2. PRRSV-CSFV-JEV可视化共检基因芯片的初步应用：应用制备的PRRSV-CSFV-JEV可视化共检基因芯片对2014年1月至2015年1月四川猪场送检的102份病料进行了检测,102份临床样本中,PRRSV的检出率为46%,与RT-PCR方法的符合率是100%；JEV的检出率为3%,与RT-PCR方法的符合率是100%；CSFV的检出率为39%,与RT-PCR方法的符合率是97.5%；存在混合感染的现象,其中PRRSV和CSFV混合感染的检出率为6.8%,PRRSV和JEV混合感染的检出率为1%,与RT-PCR方法的符合率均为100%。研究表明,PRRSV-CSFV-JEV可视化共检基因芯片可同时检测三种猪的繁殖障碍性疾病,具有特异性强、灵敏度高、高通量和可视化等优点,为基因芯片的推广应用奠定了基础。
[Abstract]:Porcine reproductive and respiratory syndrome, swine syndrome (CSF), classical swine fever (CSF) and Japanese encephalitis (je) are three common diseases causing porcine reproductive disorders. Therefore, synchronous and rapid differential diagnosis of these three diseases is of great significance. Gene chip technology is suitable for high-throughput differential diagnosis, but most of the traditional gene chip labeling technology will use fluorescein, and need expensive equipment, so it is not suitable for the extensive application of grass-roots units. In this study, the construction of PRRSV-CSFV-JEV visual visualized gene chip was carried out, and the clinical application evaluation was carried out, which provided a new gene chip technique for the diagnosis of porcine reproductive disorders. Construction of PRRSV-CSFV-JEV Microarray: PS8 gene of Y11 gene of PRRSv and C gene of JEV were designed as probes. 位 DNA was used as positive quality control gene, and the probe and quality control gene were purified by sodium ethanol acetate method. The probe gene and the positive quality control gene were sprayed onto the amino substrate in accordance with the designed array and repeated twice with the sample solution diluted to 200ng / uL by dilution to 400ng / uL, and the probe gene and the positive quality control gene were sprayed onto the amino substrate according to the designed array by spraying the probe gene and the positive quality control gene. The substrate was roasted in a oven at 80 鈩，
本文编号：2133326
[Abstract]:Porcine reproductive and respiratory syndrome, swine syndrome (CSF), classical swine fever (CSF) and Japanese encephalitis (je) are three common diseases causing porcine reproductive disorders. Therefore, synchronous and rapid differential diagnosis of these three diseases is of great significance. Gene chip technology is suitable for high-throughput differential diagnosis, but most of the traditional gene chip labeling technology will use fluorescein, and need expensive equipment, so it is not suitable for the extensive application of grass-roots units. In this study, the construction of PRRSV-CSFV-JEV visual visualized gene chip was carried out, and the clinical application evaluation was carried out, which provided a new gene chip technique for the diagnosis of porcine reproductive disorders. Construction of PRRSV-CSFV-JEV Microarray: PS8 gene of Y11 gene of PRRSv and C gene of JEV were designed as probes. 位 DNA was used as positive quality control gene, and the probe and quality control gene were purified by sodium ethanol acetate method. The probe gene and the positive quality control gene were sprayed onto the amino substrate in accordance with the designed array and repeated twice with the sample solution diluted to 200ng / uL by dilution to 400ng / uL, and the probe gene and the positive quality control gene were sprayed onto the amino substrate according to the designed array by spraying the probe gene and the positive quality control gene. The substrate was roasted in a oven at 80 鈩，
本文编号：2133326
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