Development of a multipathogen oligonucleotide microarray for detection of Bacillus anthracis
Development of a multipathogen oligonucleotide microarray for detection of Bacillus anthracis
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DOI：
10.1016/j.mcp.2005.06.004
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发表时间：
2005-10-01
影响因子：
3.3
通讯作者：
Silman, NJ
中科院分区：
文献类型：
--
作者：
Burton, JE;Oshota, J;Silman, NJ
寡核苷酸微阵列系统已被专门设计用于检测和区分炭疽芽孢杆菌与临床样品中存在的其他细菌物种。中试规模的微阵列最初包含探针来检测六种常见的细菌，并对其进行了全面评估。微阵列包括设计用于与16 S rRNA基因的可变区杂交的长寡核苷酸（50-70-mer）。还掺入了与毒力基因杂交的探针;对于B。在炭疽杆菌中，这些最初包括pag、lef、cap和vrrA（用于部分基因分型）基因。杂交条件最初被优化为使用5X SSC、0.1%SDS在50 ° C下运行16小时。在这些条件下，通过滴定染色体DNA和未标记的扩增子，然后通过杂交确定微阵列可获得的灵敏度水平，来确定微阵列的检测限。还比较了两种不同的扩增方法-基于特异性引物的PCR和随机PCR（结合标记阶段）。使用特异性PCR引物获得更高的灵敏度，然而，由于基于微阵列的检测系统的期望结果之一是其提供的高区分度，因此使用随机扩增和标记作为选择的扩增方法。使用时间过程研究杂交的长度，发现1-2小时产生最佳信号，并且比16小时孵育产生更高的信号。这些结果表明，微阵列技术可以用于诊断环境，而且，结果可以在类似的时间尺度上获得一个标准的PCR反应，但具有的优势是，没有先验知识的感染性病原体是需要检测。(C)2005爱思唯尔有限公司保留所有权利。
An oligonucleotide microarray system has been specifically designed to detect and differentiate Bacillus anthracis from other bacterial species present in clinical samples. The pilot-scale microarray initially incorporated probes to detect six common species of bacteria, which were fully evaluated. The microarray comprised long oligonucleotides (50-70-mer) designed to hybridise with the variable regions of the 16S rRNA genes. Probes which hybridised to virulence genes were also incorporated; for B. anthracis, these initially included the pag, lef, cap and vrrA (for partial genotyping) genes. Hybridisation conditions were initially optimised to be run using 5 X SSC, 0.1 % SDS' 50 degrees C for 16 h. The detection limits of the microarray were determined under these conditions by titration of chromosomal DNA and unlabelled amplicons followed by hybridisation to determine the levels of sensitivity that could be obtained with the microarray. Two different amplification methodologies were also compared-specific-primer based PCR and random PCR (with the labelling stage incorporated). Higher sensitivity was obtained using specific PCR primers, however, since one of the desired outcomes of a microarray-based detection system was the high discrimination that it offered, random amplification and labelling was used as the amplification method of choice. The length of hybridisation was investigated using a time-course, and 1-2 h was found to give optimal and higher signals than 16 h incubation. These results indicate that microarray technology can be employed in a diagnostic environment and moreover, results may be obtained in a similar time-scale to a standard PCR reaction, but with the advantage that no a priori knowledge of the infectious agent is required for detection. (C) 2005 Elsevier Ltd. All rights reserved.