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Applied and Environmental Microbiology, November 2005, p. 7236-7244, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7236-7244.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Takehiko Kenzaka, Shigeru Tamaki, Nobuyasu Yamaguchi, Katsuji Tani, and Masao Nasu*

Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamada-oka, Suita, Osaka 565-0871, Japan

Received 14 February 2005/ Accepted 10 June 2005

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 x 103 and 9.0 x 103 cells ml–1 and between undetectable and 5.1 x 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.

* Corresponding author. Mailing address: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8170. Fax: 81-6-6879-8174. E-mail: nasu{at}phs.osaka-u.ac.jp.

Applied and Environmental Microbiology, November 2005, p. 7236-7244, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7236-7244.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kenzaka, T., Tani, K., Sakotani, A., Yamaguchi, N., Nasu, M. (2007). High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level. Appl. Environ. Microbiol. 73: 3291-3299 [Abstract] [Full Text]  
  • Maruyama, F., Tani, K., Kenzaka, T., Yamaguchi, N., Nasu, M. (2006). Quantitative Determination of Free-DNA Uptake in River Bacteria at the Single-Cell Level by In Situ Rolling-Circle Amplification. Appl. Environ. Microbiol. 72: 6248-6256 [Abstract] [Full Text]  


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