Recognition of Individual Genes in Diverse Microorganisms by Cycling Primed In Situ Amplification

Abstract

Cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) was developed to recognize individual genes in a single bacterial cell. In CPRINS, the amplicon was long single-stranded DNA and thus retained within the permeabilized microbial cells. FISH with a multiply labeled fluorescent probe set enabled significant reduction in nonspecific background while maintaining high fluorescence signals of target bacteria. The ampicillin resistance gene in Escherichia coli, chloramphenicol acetyltransferase gene in different gram-negative strains, and RNA polymerase sigma factor (rpoD) gene in Aeromonas spp. could be detected under identical permeabilization conditions. After concentration of environmental freshwater samples onto polycarbonate filters and subsequent coating of filters in gelatin, no decrease in bacterial cell numbers was observed with extensive permeabilization. The detection rates of bacterioplankton in river and pond water samples by CPRINS-FISH with a universal 16S rRNA gene primer and probe set ranged from 65 to 76% of total cell counts (mean, 71%). The concentrations of cells detected by CPRINS-FISH targeting of the rpoD genes of Aeromonas sobria and A. hydrophila in the water samples varied between 2.1 × 10³ and 9.0 × 10³ cells ml⁻¹ and between undetectable and 5.1 × 10² cells ml⁻¹, respectively. These results demonstrate that CPRINS-FISH provides a high sensitivity for microscopic detection of bacteria carrying a specific gene in natural aquatic samples.