制备炭疽芽胞杆菌检测基因芯片的初步研究

Preliminary Study on DNA Microarray for Detection of Bacillus anthracis

建立制备炭疽芽胞杆菌检测基因芯片的技术 ,并探讨研制检测炭疽芽胞杆菌基因芯片的方法。酶切炭疽芽胞杆菌的毒素质粒和荚膜质粒 ,通过建立质粒DNA文库的方法获取探针 ,并打印在经过氨基化修饰的玻片上 ,制成用于炭疽芽胞杆菌检测的基因芯片。收集了 2 90个阳性克隆探针 ,制备了检测炭疽芽胞杆菌的基因芯片。提取炭疽芽胞杆菌质粒DNA与基因芯片杂交 ,经ScanArrayLite芯片阅读仪扫描得到初步的杂交荧光图像。通过分析探针的杂交信号初步筛选出 2

To develop a technique of detecting Bacillus anthracis with DNA microarray, and explore the way of preparing DNA microarray for detecting B.anthracis. B.anthracis plasmids, pX01 and pX02, were digested with Sau3AI and the resulting fragments were used to construct DNA library of pX01 and pX02 DNA microarray probes were obtained from fragments of DNA library. The positive clone fragments from DNA library verified by sequencing were used as the DNA microarray probes. A plant gene that includes GATC restriction site was used as positive control. When fluorescence labeled, it was added into the sample detecting system. Another plant gene that was nonhomologous to B.anthracis was used as negative control, and DMSO was used as blank control. A DNA microarray of detecting B.anthracis was prepared by printing the probes on a superamine modified glass slide. 290 cloned probes were collected. A DNA microarray for detecting B.anthracis was prepared. After hybridized with sample B.anthracis plasmids, pX01 and pX02, 273 cloned probes were screened by the analysis of the fluorescent intensities for further study. The high effectivity, utility and reliability of DNA microarray technique would lay a good foundation for further study on detection of B.anthracis.