Development and validation of a diagnostic microbial microarray for methanotrophs
Development and validation of a diagnostic microbial microarray for methanotrophs
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DOI：
10.1046/j.1462-2920.2003.00450.x
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发表时间：
2003-07-01
影响因子：
5.1
通讯作者：
Sessitsch, A
中科院分区：
文献类型：
--
作者：
Bodrossy, L;Stralis-Pavese, N;Sessitsch, A
DNA微阵列技术在细菌高通量检测和细菌群落结构定量评估方面的潜力已得到广泛认可，但尚未完全实现。基于现成的技术和材料，为诊断微生物微阵列的设计、生产和应用开发了一套普遍适用的技术。建立了一种针对颗粒甲烷单加氧酶（pmoA）基因的微阵列，用于甲烷氧化菌及相关功能菌的检测和定量。由59个探针组成的微阵列覆盖了这些细菌的全部已知多样性，并与一组具有代表性的现存菌株和环境克隆进行了验证。用环境样品测试了pmoA微阵列的潜力。结果与克隆文库序列分析结果吻合较好。目前，该方法可以检测到占目标菌群总数5%的优势菌。用人工PCR混合物对该体系的定量潜力进行初步测试，结果与预期结果有很好的相关性，标准差在0.4-17.2%之间。用这种方法对环境样品进行定量，需要设计由样品中菌株的近亲组成的参比混合物，目前受到环境DNA提取和通用PCR扩增固有的偏差的限制。
The potential of DNA microarray technology in high-throughput detection of bacteria and quantitative assessment of their community structures is widely acknowledged but has not been fully realised yet. A generally applicable set of techniques, based on readily available technologies and materials, was developed for the design, production and application of diagnostic microbial microarrays. A microarray targeting the particulate methane monooxygenase (pmoA ) gene was developed for the detection and quantification of methanotrophs and functionally related bacteria. A microarray consisting of a set of 59 probes that covers the whole known diversity of these bacteria was validated with a representative set of extant strains and environmental clones. The potential of the pmoA microarray was tested with environmental samples. The results were in good agreement with those of clone library sequence analyses. The approach can currently detect less dominant bacteria down to 5% of the total community targeted. Initial tests assessing the quantification potential of this system with artificial PCR mixtures showed very good correlation with the expected results with standard deviations in the range of 0.4-17.2%. Quantification of environmental samples with this method requires the design of a reference mixture consisting of very close relatives of the strains within the sample and is currently limited by biases inherent in environmental DNA extraction and universal PCR amplification.