Details

Autor(en) / Beteiligte

• Sachidanandham, Ramaiah
• Gin, Karina Yew-Hoong

Titel

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

Ist Teil von

• Biotechnology and bioengineering, 2003-04, Vol.82 (2), p.127-133

Ort / Verlag

New York: Wiley Subscription Services, Inc., A Wiley Company

Erscheinungsjahr

2003

Quelle

MEDLINE

Beschreibungen/Notizen

• An in situ PCR‐based flow cytometry method useful for monitoring the presence or absence of the β‐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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 127–133, 2003.