寡核苷酸阵列芯片快速鉴定大肠杆菌O157:H7初步研究

目的设计、制作一种微型化寡核苷酸阵列芯片，评价快速鉴定肠出血性大肠杆菌O157：H7的效果。方法多重PCR扩增大肠杆菌O157：H7七个特异性基因位点（rfbE、flicH7、intimin、Shiga-like toxins Ⅰ and Ⅱ、hemolysin A和uidA）．通过PCR反应掺入SpectrumOrang^TM-dUTP获取荧光标记的靶序列，与制备的芯片寡核苷酸探针杂交。结果寡核苷酸阵列芯片检测结果与试验预期相符，获取的杂交图分辨效果明显优于多重PCR琼脂糖凝胶电泳。结论基于玻片的寡核苷酸阵列芯片制作简便，鉴定病原菌检测细菌毒力因子快速、灵敏、特异，有良好的应用前景。

Preliminary study on detection and identification of E. Coli O157: H7 by oligonucleotide array chip

Objective To design and prepare a rapid,sensitive and specific diagnostic assays for detection of EHEC O_(157):H_7 with oligonucleotide array chip. Methods A miniature oligonucleotide arrays of gene-specific probes immobilized on glass slides was firstly fabricated for the detection of the Escherichia coli O_(157):H_7.The presence of seven gene loci(rfbE,flicH7,intimin,Shiga-like toxins I and II,hemolysin A and uidA) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by hybridization of the denatured PCR products to the bacterial strains. Results The fluorescent-labeled PCR products were prepared by incorporation of Spectrum Orange dUTP during multiplex PCR amplification and were hybridized to the array without further modification or purification.The resolution of obtained hybridization map was superior to the multiplex PCR agarose electrophoresis. Conclusion Oligonucleotide arrays analysis of microbial nucleic acid sequence is a powerful and widely applicable method for the automated identification and characterization of bacterial pathogen.