Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC)
Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC)
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DOI：
10.1186/1475-2859-7-29
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发表时间：
2008-10-13
影响因子：
6.4
通讯作者：
Gabig-Ciminska, Magdalena
中科院分区：
文献类型：
--
作者：
Basselet, Pascal;Wegrzyn, Grzegorz;Gabig-Ciminska, Magdalena
背景：利用基于 DNA 的微生物病原体分析，特别是细菌中几种毒力相关基因的同时分型，正在成为当今公共卫生的一个重要目标。结果：开发了一种样品处理程序，用于使用 DNA 芯片阵列对单个菌落进行肠出血性大肠杆菌 (EHEC) 的验证性分析，并在此进行了报道。该方案包括应用来自超声处理菌落的片段化基因组 DNA。样品处理包括前 2.5 分钟超声波处理、DNA 提取 (2x)，然后再进行 5 分钟超声波处理。因此，EHEC 验证性分析的总样品制备时间接近 10 分钟。此外，还进行了生物信息学修订，以设计针对 EHEC 相关基因的大多数保守区域的 PCR 引物和阵列探针。本研究选择了六种具有不同致病特性的菌株。最后设计并验证了并行同步检测etpC-stx1-stx2-eae基因的EHEC芯片阵列。这应该允许感测 EHEC 类型和亚型中所选序列的所有当前可访问的变体。结论：为了实施基于 DNA 芯片阵列的分析以直接检测 EHEC，在这项工作过程中建立了样品处理。然而，这种样品制备模式也可以应用于其他类型的基于 EHEC DNA 的传感系统。
Background: Exploitation of DNA-based analyses of microbial pathogens, and especially simultaneous typing of several virulence-related genes in bacteria is becoming an important objective of public health these days.Results: A procedure for sample processing for a confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony with DNA chip array was developed and is reported here. The protocol includes application of fragmented genomic DNA from ultrasonicated colonies. The sample processing comprises first 2.5 min of ultrasonic treatment, DNA extraction (2x), and afterwards additional 5 min ultrasonication. Thus, the total sample preparation time for a confirmative analysis of EHEC is nearly 10 min. Additionally, bioinformatic revisions were performed in order to design PCR primers and array probes specific to most conservative regions of the EHEC-associated genes. Six strains with distinct pathogenic properties were selected for this study. At last, the EHEC chip array for a parallel and simultaneous detection of genes etpC-stx1-stx2-eae was designed and examined. This should permit to sense all currently accessible variants of the selected sequences in EHEC types and subtypes.Conclusion: In order to implement the DNA chip array-based analysis for direct EHEC detection the sample processing was established in course of this work. However, this sample preparation mode may also be applied to other types of EHEC DNA-based sensing systems.