Flow cytometric detection of beta-D-glucuronidase gene in wild-type bacterial cells using in-situ PCR

An in situ PCR-based flow cytometry method useful for monitoring the presence or absence of the beta-D-glucuronidase gene in Escherichia coli has been developed. A single-step fixation and permeabilization procedure, which maintained cell integrity at the elevated temperatures used during thermal cycling in the presence of PCR reagents, was demonstrated. We have chosen a shorter DNA sequence of length 147 bp for the PCR. Cells subjected to in situ PCR using fluorescein-12-dUTP as a label, showed the presence of uid both in epifluorescence microscopic examination and flow cytometric analysis. Multi-parametric analysis of flow cytometric profiles revealed that the efficiency of labeling was found to be high. The potential of in situ PCR for the detection of uid in intact coliform cells was then successfully tested with a fecal coliform isolated from the coastal waters of Singapore.