DEVELOPMENT OF A DIRECT IN-SITU PCR METHOD FOR DETECTION OF SPECIFIC BACTERIA IN NATURAL ENVIRONMENTS

We applied HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate ) to direct in situ PCR for the routing detection of specific bacteria l cells at the single-cell level, PCR was performed an glass slides wi th digoxigenin-labeled dUTP. The digoxigenin-labeled PCR products were defected with alkaline phosphatase-labeled antidigoxigenin antibody a nd 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 amplificatio n 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 detec ted.