用基因芯片鉴别诊断四种水泡性疾病  

Diagnostic Genechip for Differentiation of Four Kinds of Vesicular Diseases

作　　者：秦智锋[1] 钟安清[1] 杨宝华[1] 叶奕优[1] 

机构地区：[1]深圳出入境检验检疫局动植食中心,广东深圳518010

出　　处：《中国病毒学》2003年第2期174-177,F003,共5页Virologica Sinica

基　　金：国家质量监督检验检疫总局科学研究与技术开发重大项目(K002-2000)

摘　　要：为了对临床上症状非常相似的四种水泡性动物疾病进行大批量、快速准确的检测,我们尝试用基因芯片技术来对这四种水泡性疾病进行快速鉴别诊断。用Qiaquick 96 plate Kit对扩增的39个基因目的片段进行纯化,TE调配浓度至0.36礸/礚,于GMS417点样仪上按预选设计好的布阵方式点样,然后经紫外灯照射进行紫外交联等一系列处理。用Salmon进行非特异性杂交检测芯片点样的质量。大量抽提组织中FMDV和细胞上清液中的SVDV的总RNA,随机引物反转录的同时采用随机荧光标记法,标记FMDV和SVDV。标记好的cDNA超声波随机打断后进行特异性杂交,以最佳光扫描强度进行扫描,Imagene软件扫描结果,对两者信号进行对比,结合各病毒相对应的坐标,可鉴别诊断出四种动物的水泡性疾病病毒。本方法不但快速、灵敏、准确性高,而且可实现对大批量货物的集成化检测,满足我国快速通关的要求。Genechip technology was adopted to differentiate the four animal vesicular diseases in order to distinguish them properly, rapidly and largely. Thirty-nine amplified target gene fragments were purified with Qiaquick 96 plate kit,and diluted with TE to obtain a concentration of 0.36 0.36礸/礚. A microarray was spotted with a GMS417 (pine-and-ring spotter) apparatus, after which the cDNA fragments were coupled with -NH2 on a genechip using ultraviolet cross linkage technique. Afterwards the quality of chip was confirmed by aspecific hybridization with salmon sperm. Large quantities of Foot-and-mouth disease virus (FMDV) RNA from infected tissues and Swine vesicular disease virus (SVDV) RNA from infected cell culture supernatant were extracted and, fluoromarker were used to label cDNA fragments of FMDV and SVDV when they amplified with random primers. After the labeled cDNA was randomly sheared by ultrasonic treatment, the broken pieces were subjected to specific hybridization, and then photoscanned using optimal light intensity, and the result analyzed with Imagene software. Comparison of the signals from the specimens while taking into consideration correspondent position of each virus on the genechip, allowed differential diagnoses of the four kinds of vesicular diseases. The diagnostic genechip method was rapid, sensitive and exact, rush through.

关 键 词：水泡性疾病 鉴别诊断 基因芯片 口蹄疫病毒 猪水泡病病毒 

分 类 号：S854.4[农业科学—临床兽医学]