Propidium monoazide-quantitative polymerase chain reaction for viable Escherichia coli and Pseudomonas aeruginosa detection from abundant background microflora

Abstract

Nucleic acid-based techniques represent a promising alternative to cultivation-based microbial water quality assessment methods. However, their application is hampered by their innate inability to differentiate between living and dead organisms. Propidium monoazide (PMA) treatment was proposed as an efficient approach for alleviating this limitation. In this study, we demonstrate the performance of PMA-quantitative polymerase chain reaction (qPCR) for the detection of indicator organisms (Escherichia coli and Pseudomonas aeruginosa) in a background of a highly abundant and complex microflora. Treatment with 10 μM PMA resulted in the complete or significant reduction of the false positive signal arising from the amplification of DNA from dead cells.

Keywords: Complex background microflora; E. coli; Live/dead differentiation; P. aeruginosa; Propidium monoazide; Quantitative PCR.