【摘要】：豬繁殖與呼吸綜合征(Porcine reproductive and respiratory syndrome, PRRS)、豬瘟(Classical swine fever, CSF)、流行性乙型腦炎(Japanese encephalitis, JE)是三種常見的引起豬繁殖障礙疾病,在生產(chǎn)豬場(chǎng)存在混合感染的情況較多。因此,同步快速鑒別診斷這三種病具有重要意義�；蛐酒夹g(shù)適合高通量鑒別診斷,但傳統(tǒng)的基因芯片標(biāo)記技術(shù)多數(shù)會(huì)用到熒光素,且需要價(jià)格昂貴設(shè)備,不適合基層單位的廣泛應(yīng)用。本研究開展了PRRSV-CSFV-JEV直觀可視化基因芯片的構(gòu)建,并進(jìn)行了臨床應(yīng)用評(píng)價(jià),為豬繁殖障礙病的診斷提供了新的基因芯片技術(shù)。1. PRRSV-CSFV-JEV可視化共檢基因芯片的構(gòu)建研究：選取PRRSV的Y11基因、CSFV的PS8基因和JEV的C基因設(shè)計(jì)探針,λDNA作為陽(yáng)性質(zhì)控基因,探針和質(zhì)控基因用乙醇醋酸鈉法純化,稀釋到400ng/uL,以點(diǎn)樣液等體積稀釋到200ng/uL,用噴樣的方式將探針基因和陽(yáng)性質(zhì)控基因按照設(shè)計(jì)陣列噴點(diǎn)到氨基基片上,并重復(fù)2次。點(diǎn)制完成的基片在預(yù)設(shè)為80℃的烘箱中烘焙4h,紫外交聯(lián)30min,密封、4℃保存。PRRSV-CSFV-JEV可視化共檢基因芯片的雜交流程包括靶基因的擴(kuò)增標(biāo)記、預(yù)雜交、雜交、Nanogold-Streptavidin孵育和銀染顯色判定結(jié)果。對(duì)可視化基因芯片的雜交溫度、雜交時(shí)間、Nanogold-Streptavidin的稀釋倍數(shù)、銀染顯色的時(shí)間進(jìn)行探索和優(yōu)化,并對(duì)該芯片的特異性、靈敏性、穩(wěn)定性和保存期進(jìn)行驗(yàn)證。結(jié)果表明PRRSV-CSFV-JEV可視化共檢基因芯片在48℃雜交60min,Nanogold-Streptavidin稀釋40倍,銀染時(shí)間8-10min效果最好。PRRSV-CSFV-JEV可視化共檢基因芯片特異性良好,探針之間無(wú)交叉反應(yīng),靈敏度可達(dá)16.2pg/uL,重復(fù)性良好,4℃密封至少可保存180d。2. PRRSV-CSFV-JEV可視化共檢基因芯片的初步應(yīng)用：應(yīng)用制備的PRRSV-CSFV-JEV可視化共檢基因芯片對(duì)2014年1月至2015年1月四川豬場(chǎng)送檢的102份病料進(jìn)行了檢測(cè),102份臨床樣本中,PRRSV的檢出率為46%,與RT-PCR方法的符合率是100%；JEV的檢出率為3%,與RT-PCR方法的符合率是100%；CSFV的檢出率為39%,與RT-PCR方法的符合率是97.5%；存在混合感染的現(xiàn)象,其中PRRSV和CSFV混合感染的檢出率為6.8%,PRRSV和JEV混合感染的檢出率為1%,與RT-PCR方法的符合率均為100%。研究表明,PRRSV-CSFV-JEV可視化共檢基因芯片可同時(shí)檢測(cè)三種豬的繁殖障礙性疾病,具有特異性強(qiáng)、靈敏度高、高通量和可視化等優(yōu)點(diǎn),為基因芯片的推廣應(yīng)用奠定了基礎(chǔ)。
[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，

本文編號(hào)：2133326