Water Research Institute (IRSA), National Research Council (CNR), Monterotondo St. (RM), 00015, Italy.
Cento de Recursos Naturais e Ambiente (CERENA), Faculty of Engineering, University of Porto, Porto, Portugal.
Methods Mol Biol. 2021;2246:129-140. doi: 10.1007/978-1-0716-1115-9_9.
CARD-FISH technique allows us to increase microbial cell detection compared to traditional FISH assays. Specific nonfluorescent oligonucleotide probes targeting 16S rRNA genes are employed and are chemically activated by the binding of tyramide molecules, with the latter able to generate a cascade of fluorescence signals, improving sensitivity and reducing background noise. The technique has been successfully applied for the detection of microorganisms in different environmental matrices and under different growth conditions (including those where cells are characterized by low physiological activity and low ribosome content). This chapter presents a straightforward procedure to execute CARD-FISH analysis, from sample preparation and fixation, to microscopic visualization, along with relevant technical notes.
CARD-FISH 技术可与传统 FISH 检测相比提高微生物细胞检测能力。该技术使用针对 16S rRNA 基因的特异性非荧光寡核苷酸探针，并通过与吖啶酮分子结合而被化学激活，后者能够产生荧光信号级联，从而提高灵敏度并降低背景噪声。该技术已成功应用于不同环境基质和不同生长条件（包括那些细胞生理活性和核糖体含量低的条件）下微生物的检测。本章介绍了执行 CARD-FISH 分析的简单程序，包括样品制备和固定、显微镜可视化以及相关技术说明。
