肠道传染病病原体可视化基因芯片检测技术的研究

Technology of Visualization Gene Chip in Detection of Intestinal Infectious Disease Pathogens

〔目的〕研究建立能同时检测7种肠道传染病病原体的可视化的快速、高效的基因芯片检测技术。〔方法〕针对霍乱弧菌、副溶血弧菌、沙门菌、大肠埃希菌O157:H7、金黄色葡萄球菌、轮状病毒、诺瓦克病毒(I型和II型)等7种肠道传染病病原体,设计特异引物和探针,构建芯片。以链霉亲和素包被的磁珠为信号标记分子检测芯片上的特异杂交反应,结果采用裸眼观察或普通光学设施成像保存。测试构建芯片的特异性、重复性及灵敏度等参数。〔结果〕该芯片具有较好的特异性和重复性,检测灵敏度可达25ng/μl;对27份临床粪便样本分别进行PCR和芯片检测,芯片检测结果与PCR方法检测结果一致。〔结论〕本研究建立的基于磁珠标记的可视化芯片技术,能够用于快速筛查选定的7种主要的肠道传染病病原体。该技术对于进一步开发更多指标的微生物检测方法具有很好的示范作用。

Objective To develop a rapid and efficient detection for simultaneously screening seven kinds of intestinal infectious disease pathogens by using visualization gene chip technology. Method Specific primers and probes of Vibrio Cholerae, Vibrio parahaemolyticus, SalmoneUa spp., Escherichia coli O157：H7, Staphylococcus aureus, Rotavirus, Norwalk virus （including genogroup Ⅰ and Ⅱ） were designed to establish gene chips. Streptavidin coated magnetic beads were used as markers to detect chip hybridization reaction. The results can be observed either by the a naked eye or by ordinary optical imaging facilities. The gene chip＇ s specificity, repeatability and sensitivity were also verified. Result The gene chip had a good specificity and reproducibility and its detection sensitivity was 25 ng/μl. 27 clinical fecal samples were tested by PCR and gene chip, respectively. The results of the two methods were the same. Conclusion This study suggests that the established gene chip with magnetic bead-based visualization technology can be used for rapid screening of the seven major enteric pathogens. The technology in this study has a very good role model for the further development of additional indicators of microbial detection method.