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Appl Environ Microbiol, April 1998, p. 1536-1540, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Development of a Direct In Situ PCR Method for Detection of Specific Bacteria in Natural Environments

Katsuji Tani,* Ken Kurokawa, and Masao Nasu

Department of Microbiology and Environmental Science, Faculty of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565, Japan

Received 30 September 1996/Accepted 25 January 1998

We applied HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate) to direct in situ PCR for the routine detection of specific bacterial cells at the single-cell level. PCR was performed on glass slides with digoxigenin-labeled dUTP. The digoxigenin-labeled PCR products were detected with alkaline phosphatase-labeled antidigoxigenin antibody and HNPP which was combined with Fast Red TR. A bright red fluorescent signal was produced from conversion to HNP (dephosphorylated form) by alkaline phosphatase. We used the ECOL DNA primer set for amplification of ribosomal DNA of Escherichia coli to identify cells specifically at the single-cell level in a bacterial mixture. High-contrast images were obtained under an epifluorescence microscope with in situ PCR. By image analysis, E. coli cells in polluted river water also were detected.

* Corresponding author. Mailing address: Department of Microbiology and Environmental Science, Faculty of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565, Japan. Phone: 81-6-879-8173. Fax: 81-6-879-8174. E-mail: tani{at}phs.osaka-u.ac.jp.




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