摘要
目的结合纳米放大芯片检测的专利技术,设计并制作适用于病原体快速检测的实用型基因芯片,以实现对临床常见感染病原体的快速、准确地检测和鉴定。方法分别针对各待检靶病原体特异性基因片段设计探针、构建芯片,并以纳米金为报告分子标志靶序列,通过杂交后与银的反应使得信号被放大,形成裸眼可见的褐色颗粒,从而实现芯片的检测。结果设计的探针具有很好的特异性、准确性和杂交条件的同一性;适当增加纳米金/核酸的比例能够减少标志时间,而轻微振荡明显加快标志反应的速度;在45℃杂交温度下,杂交反应盐离子浓度为0.8mol/L,杂交反应时间为8h,最后经过严格条件漂洗可以获得较佳的杂交效率,而轻微的旋转振荡则可以显著增加杂交的效率;37℃,3min×3的反应方式,可以获得较好的银染效果;本芯片系统具有较好的检测敏感性,可达到100fmol/L;与临床常规检测方法比,本芯片检测方法简单快捷,检测结果裸眼清晰可见,背景低。结论本芯片检测方法敏感性高,特异性好,操作方法简单,无需特殊设备,所设计的检测内容达到实用化水平,具有较好的临床应用前景。
OBJECTIVE A practical gene chip which aimed to detect and identify pathogens rapidly and exactly is developed on the basis of patent technology of nano-enlargement-detection. METHODS Oligonucleotide probes for the specific gene fragments of target pathogens were designed and immobilized on gene chip. Target sequences were labeled by nanogold as reporter materials. After hybridization, its results were recorded by the interaction between nanogold and silver which amplified the hybridization signal to form brown particles, which could be detected by naked eyes. RESULTS The probes designed were all of strong specificity and great reliability possessing identity of hybridization conditions. The reaction time for marking could be decreased by properly raising the ratio of nanogold and nucleic acid and the speed of labeling reaction could be fastened significantly by gentle agitation. A better hybridization results could be obtained when the samples were hybridized for 8 hours at 45℃ with 0.8 mol/L ionic strength, and then strictly rinsed. Furthermore, the hybridization efficiency could be increased remarkably by slight circumgyratation. A better chromatic effect resulted from the reaction way in 3min×3 at 37℃. The sensitivity of gene chip assays in this test could reach to 100 fmol/L. Compared with traditional detection approach, detection by the chip displayed such advantages as speediness and simplicity and the detection results could be easily recognized by naked eyes. CONCLUSIONS The chip detection technology has met the demand of design exhibiting high sensitivity, strong specificity, and easy operation without special device and showing a promising prospect.
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2007年第2期143-147,共5页
Chinese Journal of Nosocomiology
基金
国家自然科学基金课题(30300326)
军队医药卫生科研基金"十五"重点课题(01Z073)
重庆市应用基础研究计划项目(8103)
中国博士后科学基金一等资助金(2005038005)
深圳市科技计划项目(JH200504270119A)
