Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603–1 Kamitomioka, Nagaoka, Niigata, Japan.
Microbes Environ. 2010;25(1):15-21. doi: 10.1264/jsme2.me09180.
In situ detection of functional genes is informative for understanding microbial physiology. Most methods of detecting functional genes employ multiple oligonucleotides or polynucleotide probes. However, single oligonucleotide probes are superior in terms of specificity and flexibility in probe design. Here we describe the detection of a single copy functional gene, the methyl coenzyme M reductase gene, in a methanogen by two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a single oligonucleotide probe without pre-amplification of target nucleic acids. Locked-nucleic-acid-incorporated DNA probes were employed to achieve high specificity and affinity. Although problems associated with non-removable nonspecific binding of the antibody could not be overcome completely, single copy gene detection was carried out with single mismatch descriminatable specificity; however, only around 15% of cells were detected. The detection rate increased when a multiple copy gene like rrn in Escherichia coli was targeted, indicating that a certain number of target molecules are necessary to achieve a high detection rate. Although possible applications of this technique to environmental samples remain restricted, the results presented the potential of gene detection by FISH with single oligonucleotide probes.
原位检测功能基因对于了解微生物生理学很有帮助。大多数检测功能基因的方法都采用多个寡核苷酸或多核苷酸探针。然而，单链寡核苷酸探针在探针设计的特异性和灵活性方面具有优势。在这里，我们描述了通过双通酪胺信号放大-荧光原位杂交（two-pass TSA-FISH），使用单个寡核苷酸探针，无需目标核酸的预扩增，对产甲烷菌中的单个拷贝功能基因（甲基辅酶 M 还原酶基因）进行检测。采用带有锁核酸的 DNA 探针以实现高特异性和亲和力。尽管无法完全克服抗体不可去除的非特异性结合问题，但通过单碱基错配可识别特异性进行单拷贝基因检测；然而，只有约 15%的细胞被检测到。当靶向大肠杆菌中的 rrn 等多拷贝基因时，检测率会增加，这表明需要一定数量的靶分子才能实现高检测率。尽管该技术在环境样本中的可能应用仍受到限制，但结果表明了通过 FISH 用单链寡核苷酸探针进行基因检测的潜力。
