DEVELOPMENT OF A NOVEL SENSITIVE FISH FOR DETECTION OF FUNCTIONAL GENE COMBINED WITH CLICK CHEMISTRY AND HCR-FISH

Abstract

 This study aimed to develop a highly sensitive method for visualizing the functional genes of microorganisms possessing endogenous peroxidase activity. We combined Hybridization Chain Reaction (HCR)-fluorescence in situ hybridization (FISH), a high-sensitivity FISH method that does not rely on enzymatic reactions, with click chemistry capable of specific binding of alkyne-azide. First, Escherichia coli containing plasmids with functional genes as a model microorganism was used to optimize the concentration and reaction time of click chemistry. Our results revealed that extending of the reaction time of click chemistry was more effective than increasing the concentration of Cu²⁺ for specific detection. Secondly, we attempted to visually detect the functional genes of anammox bacteria in a mixed sample of sludge from a bioreactor containing anammox bacteria and a pure culture of methane-producing archaea. As a result, only anammox bacteria were detected, with methane-producing archaea remaining undetected. These results indicate that our method allows for the visualization of functional genes of microorganisms in the environmental samples without being influenced by endogenous peroxidase activity. Moreover, it can be combined with microbial sorting devices for whole-genome analysis to elucidate the metabolic pathways of specific uncultured microorganisms.