Fluorescence in situ hybridization (FISH) can provide information on the morphology, spatial arrangement, and local environment of individual cells enabling the investigation of intact microbial communities. GeneFISH uses polynucleotide probes and enzymatic signal amplification to detect genes that are present in low copy numbers. Previously, this technique has only been applied in a small number of closely related organisms. However, many important functional genes, such as those involved in xenobiotic degradation or pathogenesis, are present in diverse microbial strains.

Here, we present a geneFISH method for the detection of the functional gene etnC, which encodes the alpha subunit of an alkene monooxygenase used by aerobic ethene and vinyl chloride oxidizing bacteria (etheneotrophs). The probe concentration was optimized and found to be 100 pg/μl, similar to previous geneFISH reports. Permeabilization was necessary for successful geneFISH labeling of Mycobacteria; sequential treatment with lysozyme and achromopeptidase was the most effective treatment. This method was able to detect etnC in several organisms including Mycobacteria and Nocardioides, demonstrating for the first time that a single geneFISH probe can detect a variety of alleles (>80% sequence similarity) across multiple species.

Detection of etnC with geneFISH has practical applications for bioremediation. This method can be readily adapted for other functional genes and has broad applications for investigating microbial communities in natural and engineered systems.

荧光原位杂交 (FISH) 可以提供有关单个细胞的形态、空间排列和局部环境的信息，从而能够研究完整的微生物群落。GeneFISH 使用多核苷酸探针和酶促信号放大来检测以低拷贝数存在的基因。此前，该技术仅应用于少数密切相关的生物。然而，许多重要的功能基因，例如那些参与外源性降解或发病机制的基因，存在于不同的微生物菌株中。

在这里，我们提出了一种用于检测功能基因etnC的geneFISH 方法，该基因编码需氧乙烯和氯乙烯氧化细菌（嗜氧菌）使用的烯烃单加氧酶的α 亚基。探针浓度经过优化，发现为 100 pg/μl，与之前的geneFISH 报告相似。透化对于分枝杆菌的成功基因FISH标记是必要的；用溶菌酶和去色肽酶序贯治疗是最有效的治疗方法。该方法能够检测包括分枝杆菌和诺卡氏菌在内的多种生物体中的etnC，首次证明单个geneFISH 探针可以检测多个物种的多种等位基因（>80% 序列相似性）。

用geneFISH检测etnC在生物修复方面具有实际应用。这种方法可以很容易地适用于其他功能基因，并且在研究自然和工程系统中的微生物群落方面具有广泛的应用。