埃博拉病毒检测基因芯片的制备初探

    夏文英1,2，郑夔1，黄吉城1，伊怀文1,2

    1.广东出入境检验检疫局技术中心，广东 广州510700；2.中山大学公共卫生学院

    摘要：目的制备检测埃博拉病毒（EBOV）的基因芯片，并探索利用EBOV质粒对其进行初步验证。方法 设计20条特异性70mer寡核苷酸探针，点样制备成9×8的芯片阵列。采用phi 29DNA聚合酶多重置换扩增（MDA）和荧光标记PCR技术，将标记好的样本与经过预杂交的芯片杂交、洗脱、扫描、最终进行数据分析。结果埃博拉病毒质粒与芯片上的探针杂交后，经扫描出现特异性的荧光信号，部分信号值已达到饱和，阳性对照有明显的杂交信号，而阴性对照和空白对照则呈弱或无荧光信号。芯片可检测的灵敏度达到104拷贝数。结论本实验表明该芯片具有较高的特异性、灵敏度和重复性，可为埃博拉病毒的检测提供一种有效的方法，为进一步建立埃博拉病毒的高通量筛查和鉴定技术平台提供了实验依据。

    关键词：埃博拉病毒；基因芯片；检测；制备

    中图分类号：R183.9　文献标识码：B

    Preliminary preparation of microarray fordetecting Ebola virus

    XIA Wen-ying*, ZHENG Kui, HUANHG Ji-cheng, YIHuai-wen

    *Guangdong Entry-exit Inspection andQuarantine Technology, Guangzhou, Guangdong 510700,China

    Abstract:   Objective  To prepare microarray for detecting Ebola virus, and use EBOVplasmid to get the preliminary validation.  Methods    According to the probe design principles,20 specific 70 mer oligonucleotide probes were designed, the probeswere spotted onto the slides, forming the array of 9 × 8. Use thephi 29 DNA polymerase MDA and fluorescent labeling PCR technologyfor the sample preparation. After that, the labeled sample washybridized with pre-hybridized microarray, then the slide waswashed, scanned and finally the data wereanalyzed.    Results     Afterthe hybridization of Ebola virus plasmid and the chip, by scanningit showed specific fluorescence signals, and some signal value hadreached saturation, and the positive signals were obvious, whilethe negative control and blank control had weak or no fluorescencesignal. The sensitivity of the chip could reach 104copies.   Conclusion    The study showedthat the chip had a good specificity, sensitivity andrepeatability, which provided an effective way for the detection ofEbola virus and provided an experimental basis for furtherestablishment high-throughput screening and identificationtechnology platform of Ebola virus.

    Key words:    Ebola virus;Gene chip; Detection; Preparation

    《中国国境卫生检疫杂志》