High-Gain Bacterial RNA Imaging via High-Density Protein Fluorescence In Situ Hybridization

Abstract

Fluorescence in situ hybridization (FISH) is important for bacterial genotypic analysis; however, the limited number of labels per probe results in insufficient signal gain, compromising the detection of low-abundance RNAs. Here, we present high-density protein FISH (HD-FISH) for high-gain bacterial RNA imaging. Instead of fluorophore-modified probes, this method employs a fluorescently tagged, catalytically inactive ribonuclease H1 for multi-site labeling of target RNAs. Compared to traditional FISH and hybridization chain reaction-based FISH, HD-FISH suppresses background noise while enhancing positive signals, achieving 7.43-fold and 1.89-fold increases in signal gain, respectively, thereby enabling high-gain imaging. We showed that HD-FISH can precisely quantify quinolone-resistant Salmonella enterica strains within complex microbial communities, with a recovery rate of 97.78−131.40%, and allows us to detect and identify quinolone-resistant bacteria in fecal samples. The method provides a robust tool for profiling bacterial genotypes at the single-cell level, facilitating the elucidation of microbial heterogeneity and its roles in complex populations.