Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction).
Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction).
复制标题
DOI：
10.1186/1471-2180-2-27
复制
发表时间：
2002-09-20
期刊：
影响因子：
4.2
通讯作者：
De Bellis G
中科院分区：
文献类型：
--
作者：
Busti E;Bordoni R;Castiglioni B;Monciardini P;Sosio M;Donadio S;Consolandi C;Rossi Bernardi L;Battaglia C;De Bellis G
细菌16S rRNA基因的PCR扩增为细菌群落取样提供了最全面、最灵活的手段。尽管这种方法不适合大规模筛选，但对这些克隆片段的序列分析可以提供对微生物种群的定性和定量了解。其他方法，如变性梯度凝胶电泳，异双工或末端限制性片段分析是快速的，因此适用于现场规模的实验。微阵列技术是这些分析工具最近的一个新成员。在这里，我们提出了我们的结果，使用通用DNA微阵列方法作为细菌鉴别的分析工具。所提出的程序是基于DNA连接反应的性质，需要设计两个探针特定于每个目标序列。一个寡核苷酸带有荧光标记，另一个带有唯一序列（ZipCode或互补ZipCode），用于识别连接产物。在适当模板（PCR扩增的16s rRNA基因片段）存在的情况下获得的结扎片段包含荧光标记或独特序列，因此被定位到微阵列上ZipCode序列被发现的位置。因此，这种阵列是“通用的”，与特定的分子分析无关。在这里，我们提出了针对某些细菌群的探针设计及其在细菌诊断中的应用。结合使用选择性探针、结扎反应和通用阵列方法，产生了一种对细菌具有良好辨别能力的分析方法。
PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria.
登录
查看更多内容
影响因子：
4.4
作者：
Rudi, K;Skulberg, OM;Jakobsen, KS
通讯作者：
Jakobsen, KS
影响因子：
4.4
作者：
Guschin, DY;Mobarry, BK;Mirzabekov, AD
通讯作者：
Mirzabekov, AD
影响因子：
5.6
作者：
Gerry, NP;Witowski, NE;Barany, F
通讯作者：
Barany, F
影响因子：
3.2
作者：
WEISBURG, WG;BARNS, SM;LANE, DJ
通讯作者：
LANE, DJ
影响因子：
46.9
作者：
Favis, R;Day, JP;Barany, F
通讯作者：
Barany, F