Detection of Viable Escherichia coli in Environmental Water Using a Combined Propidium Monoazide Staining-real-time PCR

Introduction: Escherichia coli, as a principal fecal indicator bacterium, is used to monitor water quality world-wide.  Real-time PCR (qPCR) is a promising way to achieve a rapid and sensitive detection of E. coli in water samples.  The ability to detect only viable E. coli cells specifically, provides a more accurate reflection of water quality and safety. 

Purpose: The objective of this study was to test a new self-designed primer set targeting the ycjM gene of E. coli in a propidium monoazide (PMA)-qPCR assay, and to investigate its specificity and efficiency in detecting only viable E. coli in water.

Methods: Specificity of the ycjM primer set was checked using eight different E. coli strains, including E. coli ATCC 25922, and six other strains from environmental sources, as well as S. dysenteriae, S. flexneri and S. sonnei.  A freshly grown culture of E. coli DR23 (deer isolate) was spiked into lake water and serially diluted to 10-10⁷ CFU/ml.  E. coli counts were determined by the U.S. Environmental Protection Agency (EPA) Method 1603 and samples were treated with PMA, followed by DNA isolation and amplification by SYBR^® Green q-PCR targeting the ycjM gene primers.

Results: With the use of the ycjM primer set, all eight E. coli spp. and none of the Shigella spp. tested were detected by the PMA-qPCR.  As low as 10³ CFU/ml of viable E. coli DR23 in lake water could be detected by this method. 

Significance: Compared with the EPA standard culture-based method, PMA-qPCR targeting the ycjM gene demonstrates a very specific and efficient alternative way to detect only viable E. coli in environmental waters.