禽流感病毒等RNA病毒鉴别基因芯片的制备  

Preparation for discrimination microarray of RNA viruses

作　　者：田丽娜[1] 王秀荣[1] 杨忠苹[1] 石霖[1] 陶启蒙[1] 包红梅[1] 李雁冰[1] 陈化兰[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所农业部动物流感重点开放实验室

出　　处：《中国兽医科学》2008年第8期685-690,共6页Chinese Veterinary Science

基　　金：中俄国际合作项目(2007DFR30360)

摘　　要：将克隆和鉴定的AIV的HA、NA、M、NS、NP基因以及NDV、IBDV、IBV的保守基因片段作为探针，同时以克隆的GAPDH基因作为阳性对照，利用芯片点样系统spotArray TM24将探针与阳性对照、阴性对照和空白对照按照设计好的阵列点在基片上，制备了AIV等RNA病毒诊断芯片，并从点样条件和杂交条件两个方面进行了优化。结果显示，探针浓度在300～1200μg／mL时点样，Cy3-dUTP终浓度为0．05μmol／mL，杂交温度在42℃，杂交时间在8～12h时，杂交信号比较理想。该方法对20份已鉴定的H5N1亚型禽流感病毒的检出率为100％（20／20）；10份现地送检的疑似AIV样品的检出率为70％（7／10），检测结果与RT—PCR和鸡胚传代分离鉴定的符合率为100％。结果表明，制备的DNA芯片可有效诊断上述病毒。HA, NA, M, NS, NP genes of avian influenza virus（AIV） and Newcastle disease virus （NDV） ,infectious bursal disease virus （IBDV）, and infectious bronchitis virus （IBV） conservative gene fragments were cloned and identified as probes, while GAPDH gene was cloned as positive control. Probes, positive and negative controls were dotted on amino slides by microarray dotting system of spotArrayTM24, for preparation of discrimination microarray of RNA viruses. The reaction conditions were optimized from dotting to hybridizing. The results indicated that the signal was more idealistic when dotting concentrations were 300μg/mL to 1 200 μg/mL,final concentration of Cy3-dUTP was 0.05 μmol/mL,hybridizing temperature was 42 ℃ ,and hybridizing time was 8 h to 12 h. Detection rate for the identified 20 avian influenza virus HSN1 subtype samples was 100%（20/20） and detection rate for 10 suspected AIV samples was 70% （7/10） by the prepared microarray,which were completely in coincidence with those by RT-PCR and the chicken embryo passage separating method. The results showed that the prepared DNA microarray provided an effective method for diagnosing the above RNA viruses.

关 键 词：禽流感病毒 基因芯片 鉴别诊断 优化 

分 类 号：S852.64[农业科学—基础兽医学]