PMID: 8593955Dec 1, 1995Paper

Flow cytometric detection of specific genes in genetically modified bacteria using in situ polymerase chain reaction

FEMS Microbiology Letters
J PorterC Edwards

Abstract

Use of the polymerase chain reaction, coupled with flow cytometry, to detect a plasmid encoded xylE gene sequence in intact cells of Escherichia coli and Pseudomonas putida was investigated. Optimal incorporation of fluorescently labelled dUTP into a full length PCR product required substitution at a level of 2:3 dUTP:dTTP. Formaldehyde fixed cells of both species were counted before and after thermal cycling. Sufficient numbers of cells remained intact for subsequent detection using microscopy and flow cytometry but light scatter properties were altered. Intact cell suspensions of both species containing plasmid pLV1013 were subjected to thermal cycling with fluorescent dUTP in the reaction mix. Subsequent analysis by flow cytometry allowed detection of a fluorescent PCR product associated with cells. Control samples (without the plasmid) showed only background fluorescence. This method demonstrates the potential for applying DNA amplification methods for sensitive detection of specific sequences localized inside intact bacterial cells.

References

Oct 1, 1995·The Journal of Applied Bacteriology·J PorterR W Pickup
Mar 1, 1993·Applied and Environmental Microbiology·J A MorganR W Pickup

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Citations

Apr 1, 1997·Molecular Biotechnology·J K Jansson, J I Prosser
Jan 5, 2002·Applied and Environmental Microbiology·Katsuji TaniMasao Nasu
Aug 7, 2003·Applied and Environmental Microbiology·Fumito MaruyamaMasao Nasu
Sep 23, 2000·Journal of Microbiological Methods·J Porter, R W Pickup
Oct 16, 1999·Journal of Microbiological Methods·A Vaid, A H Bishop
May 19, 2009·Systematic and Applied Microbiology·Christian LaflammeCaroline Duchaine
Mar 27, 2010·FEMS Microbiology Reviews·Susann Müller, Gerhard Nebe-von-Caron
Feb 14, 2003·Biotechnology and Bioengineering·Ramaiah Sachidanandham, Karina Yew-Hoong Gin
May 29, 1999·Letters in Applied Microbiology·K KurokawaM Nasu
Sep 5, 2008·Current Protocols in Cytometry·Jonathan Porter
Apr 12, 2002·International Journal for Parasitology·Paul T MonisChristopher P Saint
Nov 15, 1997·Applied and Environmental Microbiology·G WallnerR Amann

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