Portable system for microbial sample preparation and oligonucleotide microarray analysis
Portable system for microbial sample preparation and oligonucleotide microarray analysis
复制标题
DOI：
10.1128/aem.67.2.922-928.2001
复制
发表时间：
2001-02-01
影响因子：
4.4
通讯作者：
Mirzabekov, AD
中科院分区：
文献类型：
--
作者：
Bavykin, SG;Akowski, JP;Mirzabekov, AD
我们开发了一种用于微生物鉴定的三组分系统，包括（i）用于连续DNA和RNA分离、分级分离、片段化、荧光标记以及去除过量游离标记和短寡核苷酸的通用的双柱操作的二氧化硅微柱;（ii）用于16 S rRNA鉴定的固定化寡核苷酸探针的微阵列;和（iii）便携式电池供电装置，用于对荧光标记的RNA片段与阵列的杂交进行成像。微柱结合了核酸分离的硫氰酸胍方法和新开发的基于羟基自由基的DNA和RNA标记和片段化技术。DNA和RNA也可以通过双链和单链形式的核酸与二氧化硅的差异结合来分级分离。该过程包括用不同溶液顺序洗涤柱。不需要真空过滤步骤、苯酚提取或离心。杂交后，整体荧光图案被捕获为数字图像或作为宝丽来照片。该三组分系统用于区分大肠杆菌、枯草芽孢杆菌、苏云金芽孢杆菌和人HL 60细胞。该程序是快速的：从全细胞开始，大约需要25分钟获得标记的DNA和RNA样品，另外需要25分钟杂交并使用固定图像分析系统或便携式成像仪获得微阵列图像。
We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica, The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.