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 29 DNA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。
Objective To prepare microarray for detecting Ebola virus, and use EBOV plasmid to get the preliminary validation. Methods According to the probe design principles, 20 specific 70 mer oligonucleotide probes were designed, the probes were spotted onto the slides, forming the array of 9 × 8. Use the phi 29 DNA polymerase MDA and fluorescent labeling PCR technology for the sample preparation. After that, the labeled sample was hybridized with pre-hybridized microarray, then the slide was washed, scanned and finally the data were analyzed. Results After the hybridization of Ebola virus plasmid and the chip, by scanning it showed specific fluorescence signals, and some signal value had reached saturation, and the positive signals were obvious, while the negative control and blank control had weak or no fluorescence signal. The sensitivity of the chip could reach 10 4 copies. Conclusion The study showed that the chip had a good specificity, sensitivity and repeatability, which provided an effective way for the detection of Ebola virus and provided an experimental basis for further establishment high-throughput screening and identification technology platform of Ebola virus.
出处
《中国国境卫生检疫杂志》
CAS
2011年第3期150-154,共5页
Chinese Journal of Frontier Health and Quarantine
基金
国家质检总局科技基金项目(2008IK198
2008IK256)