Steinlein L M, Crawford J T
Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, Georgia 30333, USA.
J Clin Microbiol. 2001 Mar;39(3):871-8. doi: 10.1128/JCM.39.3.871-878.2001.
We have developed an amplification-based reverse dot blot assay for the detection of specific sites of insertion of the Mycobacterium tuberculosis insertion sequence IS6110. In this assay, a set of biotin-labeled amplicons representing the various copies of IS6110 and their flanking sequences is generated by linker-mediated PCR. The amplicons are then hybridized to immobilized oligonucleotide probes that are specific for known IS6110 insertion sites. The method was evaluated using an array of oligonucleotide probes corresponding to IS6110 insertion sites from M. tuberculosis strains CDC1551, Erdman, and H37Rv, and multidrug-resistant strain W. A set of 72 DNA samples from 60 M. tuberculosis clinical isolates was analyzed for the presence or absence of these insertion sites, and the assay was found to be highly reproducible. This method of identifying insertion sites has been named "insite" and can be used for the genotyping of M. tuberculosis complex strains based on IS6110 insertion site profiles.
我们开发了一种基于扩增的反向斑点杂交检测方法，用于检测结核分枝杆菌插入序列IS6110的特定插入位点。在该检测方法中，通过接头介导的PCR产生一组生物素标记的扩增子，这些扩增子代表IS6110的各种拷贝及其侧翼序列。然后将扩增子与固定的寡核苷酸探针杂交，这些探针针对已知的IS6110插入位点具有特异性。使用一系列对应于结核分枝杆菌菌株CDC1551、 Erdman、H37Rv和多重耐药菌株W的IS6110插入位点的寡核苷酸探针评估该方法。对来自60株结核分枝杆菌临床分离株的72份DNA样本分析这些插入位点的有无，发现该检测方法具有高度可重复性。这种鉴定插入位点的方法被命名为“insite”，可用于基于IS6110插入位点图谱对结核分枝杆菌复合群菌株进行基因分型。
