In situ detection of genes in bacterial cells using flourescent dye-labeled polynucleotide probes (RING-FISH): Improvements of specificity and permeabilization as well as its application for environmental studies

最近，我们首次成功地通过Ring-FISH(荧光原位杂交识别单个基因)在细胞水平上显示了细菌基因。该方法基于一种改进的使用基因特异性多核苷酸转录本探针的细胞原位杂交程序。这些基因的存在是通过探针赋予细胞的荧光来指示的。典型的晕状细胞荧光通常通过激光扫描显微镜观察到。使用rRNA靶向寡核苷酸探针的传统鱼类受益于自然靶向扩增，而单个或寡拷贝基因的检测基本上依赖于密集的信号放大。如最近所示，后者是通过分子间探针网络的形成来实现的。虽然这种新的、强大的和有吸引力的技术的普遍适用性已经被证明，但在该方法可以用于环境研究之前，还需要进一步的必要改进。鉴于到目前为止，该方法只适用于一小部分基因和少数革兰氏阴性物种，因此它必须适应更广泛的基因(特别是nifH)和细菌(特别是革兰氏阳性菌)。关于探针的特异性，由于需要多核苷酸探针而产生的限制。必须开发由多个单位的基因特异性寡核苷酸组成的合成结构，以确保更高的特异性。在这种背景下，需要通过综合探针构建来解决网络形成的问题。此外，为了更好地获得多核苷酸探针，革兰氏阳性细菌的通透性必须得到改善。将进行环境研究，将环鱼分类的nifH基因分配给由常规rRNA鱼鉴定的微生物细胞。

Recently, we could for the first time successfully visualize bacterial genes at cellular level by RING-FISH (Recognition of Individual Genes by Fluorescent In Situ Hybridization). The method is based upon a modified in situ cell hybridization procedure using gene specific polynucleotide transcript probes. The presence of the respective genes is indicated by probe conferred cell fluorescence. The typically halo-like cell fluorescence is usually visualized by laser scanning microscopy. Whereas conventional FISH applying rRNA-targeted oligonucleotide probes benefits from natural target amplification, the detection of single or oligocopy genes essentially depends upon intensive signal amplification. As shown recently, the latter is conferred by inter-molecular probe network formation. Although the general applicability of this new, powerful and attractive technique has been shown, further essential improvements are necessary before the method can be used for environmental studies. Given that the approach has been applied for a small selection of genes and few gram-negative species thus far, it has to be adapted for a broader spectrum of genes (especially nifH) and bacteria (especially gram-positives). Concerning probe specificity limitations result from the need for polynucleotide probes. Synthetic constructs comprising multiple units of gene specific oligonucleotides have to be developed to ensure higher specificity. In this context, major efforts are needed to solve the problem of network formation by synthetic probe constructs. Moreover, the permeabilization of gram-positive bacteria has to be improved for a better access of polynucleotide probes. Environmental studies will be performed concerning the assignment of nifH genes classified by RING-FISH to microbial cells identified by conventional rRNA FISH.