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.
Sachidanandham
Ramaiah
R
Tropical Marine Science Institute, 14 Kent Ridge Road, National University of Singapore, Singapore 119223. tmsrs@nus.edu.sg
Gin
Karina Yew-Hoong
KY
D004755:Enterobacteriaceae / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005240:Feasibility Studies; D005243:Feces / Q000382:microbiology; D005434:Flow Cytometry / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012623:Seawater / Q000382:microbiology; D012680:Sensitivity and Specificity
D005966:Glucuronidase