In situ reverse transcription for the specific detection of bacteria and protozoa

In situ hybridization experiments with oligonucleotide probes directed against the 16S and 18S rRNA molecules have been used successfully to identify specific organisms in mixed microbial populations. However, there are limitations in applying these techniques to environmental samples. In the present study we have examined the possibility of using in situ reverse transcription as an alternative to hybridization methods for the rapid detection of Escherichia coli and the waterborne parasite Cryptosporidium parvum. Following fixation and permeabilization of the cells, extension reactions were performed with species-specific primers, AMV reverse transcriptase and either cy3-AP3-dUTP or fluorescein-11-dUTP at 45 degrees C for 45 min. The cells or oocysts were then filtered onto Costar metallic membrane filters and images captured with a CCD camera. The results have shown that this technique can successfully detect E. coli cells and C. parvum oocysts in under 2 h.