Detection in coal tar waste-contaminated groundwater of mRNA transcripts related to naphthalene dioxygenase by fluorescent in situ hybridization with tyramide signal amplification
Detection in coal tar waste-contaminated groundwater of mRNA transcripts related to naphthalene dioxygenase by fluorescent in situ hybridization with tyramide signal amplification
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DOI：
10.1016/s0167-7012(02)00015-5
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发表时间：
2002-06-01
影响因子：
2.2
通讯作者：
Madsen, EL
中科院分区：
文献类型：
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作者：
Bakermans, C;Madsen, EL
理想的生态代谢活性测定将应用于立即固定在现场的自然发生的微生物群体，并且该测定将集中于指示动态生物地球化学过程的细胞内参数。在这项研究中。荧光原位杂交 (FISH) 结合酪酰胺信号放大 (TSA) 检测细菌的细胞内 mRNA。通过使用滨松彩色冷冻 CCD 相机和延长曝光时间，提高了检测灵敏度。恶臭假单胞菌 NCIB 9816-4（一种模型萘降解细菌）用于改进方案。探针 Ac627BR 开发用于检测萘双加氧酶 (nahAc) mRNA 转录本。仅诱导细胞显示出与探针 Ac627BR 的阳性杂交。杂交前通过 RNase A 或 DNase I 消化样品来验证结果。当应用于现场固定的地下水样品时，萘双加氧酶 mRNA 探针会对受污染地下水中存在的细胞子集（大约 1%）发出荧光。该方法代表了实现环境微生物学长期目标之一的进展：同时确定单个微生物在其居住的原位土壤、水或沉积物中的身份、活性和生物地球化学影响。 (C) 2002 Elsevier Science B.V 保留所有权利。
The ideal ecological metabolic activity assay would be applied to naturally occurring microbial populations immediately fixed in the field, and the assay would focus upon intracellular parameters indicative of a dynamic biogeochemical process. In this study. fluorescent in situ hybridization (FISH) with tyramide signal amplification (TSA) detected intracellular mRNA in bacteria. Detection sensitivity was enhanced by using a Hamamatsu color chilled CCD camera and extended exposure times. Pseudomonas putida NCIB 9816-4, a model naphthalene degrading bacterium, was used to refine the protocol. Probe Ac627BR was developed for detecting naphthalene dioxygenase (nahAc) mRNA transcripts. Only induced cells showed positive hybridization to probe Ac627BR. Results were verified by RNase A or DNase I digestion of samples prior to hybridization. When applied to field-fixed groundwater samples, the naphthalene dioxygenase mRNA probe conferred fluorescence on a subset (similar to1%) of the cells present in the contaminated groundwater. This methodology represents progress towards achieving one of the longstanding goals of environmental microbiology: to simultaneously ascertain the identity, activity, and biogeochemical impact of individual microorganisms in situ-in soil, water, or sediment where they dwell. (C) 2002 Elsevier Science B.V All rights reserved.