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

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.