结合不对称PCR技术构建鸡NDV-IBV-ILTV联合检测基因芯片  

Construction of Gene Chip for Detecting NDV- IBV- ILTV of Chicken(Gallus gallus) with Asymmetric PCR

作　　者：杨国淋 赵松[1] 尹人杰 黄小波[1] 马锐[1] 张仙[1] 曹三杰[1] 文翼平[1] 伍锐[1] 文心田[1] 

机构地区：[1]四川农业大学动物医学院/动物传染病与基因芯片实验室,成都611130 [2]崇州市农村发展局,成都611200

出　　处：《农业生物技术学报》2016年第1期142-150,共9页Journal of Agricultural Biotechnology

基　　金：公益性行业(农业)科研专项项目(No.201203056)

摘　　要：基因芯片(gene chip)是依据核酸杂交原理发展的一种生物新技术,在生命科学研究领域具有重要的应用价值。本研究将不对称PCR和基因芯片两种技术相结合,构建了同步检测鸡(Gallus gallus)传染性喉气管炎病毒(Infectious laryngotracheitis virus,ILTV)、新城疫病毒(Newcastle disease virus,NDV)、传染性支气管炎病毒(Infectious bronchitis virus,IBV)的共检基因芯片。分别选取ILTV的胸苷激酶(thymidine kinase,TK)和糖蛋白B(glycoproteins B,g B)基因、NDV的融合蛋白(fusion,F)和血凝素-神经氨酸酶蛋白(haemagglutinin-neuraminidase,HN)基因以及IBV的膜蛋白(membrane,M)和核衣壳(nucleocapsid,N)基因设计引物,从重组质粒菌中扩增制备探针基因,用乙醇沉淀法纯化后点制于氨基修饰的载玻片上,制备基因芯片;靶基因用cy3标记引物,进行不对称PCR扩增,扩增的荧光标记单链产物与芯片杂交。不对称PCR结果显示,当限制性引物与非限制性引物浓度比例在1∶10时ILTV-TK、NDV-HN和IBV-N的单链产物增加最多,当浓度比在1∶20时,ILTV-g B、NDV-F和IBV-M的单链产物增加最多;相应的标记样品与3种病毒检测芯片杂交后,均出现较强的杂交信号,而阴性对照检测不到荧光信号,灵敏性实验表明,当DNA浓度为1.8×104拷贝时杂交仍为阳性。本研究构建的诊断基因芯片对12份临床样品进行初步应用检测,与PCR检测技术检出率基本一致。本实验所建立的联合检测基因芯片能够快速、准确、高通量的诊断NDV-IBV-ILTV,可以应用于集约化养殖业中对多种鸡疫病病毒的检测。Gene chip is a new biological technology based on the principle of nucleic acid hybridization,which shows important applications in the field of life science research. A method of simultaneously detecting Infectious laryngotracheitis virus（ILTV）, Newcastle disease virus（NDV） and Infectious bronchitis virus（IBV）using DNA microarray and asymmetric PCR methods was developed. Primers based on specific conservativeDNA fragments of the recombinant plasmid containing thymidine kinase（TK） and glycoproteins B（g B） genes of ILTV, fusion（F） and haemagglutinin-neuraminidase（HN） genes of NDV, membrane（M） and nucleocapsid（N） gene of IBV were designed and probe genes were amplified. Probes were purified using ethanol precipitation method and spotted on the amido- coating- glass slides to form a DNA microarray. Target genes were synthesized using fluorescent cy3- labeled primer by asymmetric PCR method and hybridized to microarray. Result of asymmetric PCR showed that single stranded of ILTV- TK, NDV- HN and IBV- N increased the most of all when concentration ratio of the restrictive and non- restrictive primer at 1∶10, and single product of ILTV-g B, NDV-F and IBV-M increased the most of all when the concentration ratio at 1∶20.The hybridization results showed the microarrays specific to the 3 poultry viruses could specifically hybridize with the corresponding sample, while any fluorescent signals could not be seen in negative controls. The sensitivity test showed that the concentration of DNA with 1.8×104copies was still positive. Compared with the previous chip, hybridization efficiency and the hybridization signals increased significantly. Detection of12 clinical samples, the results showed that the detection rate of DNA microarray and PCR method was consistent. This study showed that the developed microarray can simultaneously diagnosis 3 poultry disease of NDV- IBV- ILTV with fast, accurately and high- throughput, which can be applied to the detection a variety disease virus of chic

关 键 词：鸡 新城疫病毒(NDV) 传染性喉气管炎病毒(ILTV) 传染性支气管炎病毒(IBV) 不对称PCR 芯片构建 

分 类 号：S855.3[农业科学—临床兽医学]