Detection of viable Escherichia coli in environmental water using combined propidium monoazide staining and quantitative PCR

Water Research
Yuan YuanAzlin Mustapha

Abstract

The objectives of this study were to specifically detect viable Escherichia coli in environmental waters by targeting the ycjM gene in a propidium monoazide (PMA)-qPCR assay. PMA is a viability dye that can inhibit the amplification of DNA from dead cells, thus allowing for the detection and quantification of only viable cells. The ycjM primers were used to target E. coli that directly originated from the feces of warm blooded animals, and avoid false positive detection caused by "naturalized" E. coli that can exist in the environment. In this study, tap water and environmental waters were inoculated with E. coli isolated from animal feces. Following cell collection, samples were treated with PMA, followed by DNA isolation and qPCR detection. For pure cultures, 5 μM PMA with a 10-min light exposure was efficient at inhibiting the amplification of DNA from 105 CFU/mL dead E. coli cells, with a detection limit of 102 CFU/100 mL viable cells. For tap and environmental waters collected in the winter, a 10 μM PMA was required and as low as 103 CFU/100 mL viable cells could be detected in the presence of 105 CFU/100 mL dead cells. For water samples collected during the summer, 102 CFU/10 mL viable cells could be detected in the prese...Continue Reading

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Citations

Jul 26, 2019·Environmental Microbiology Reports·Lisa GorskiMichael B Cooley
Dec 14, 2019·International Journal of Food Microbiology·Marilia MiottoSalina Parveen
Jul 24, 2021·Biotechnology and Applied Biochemistry·Sebnem SeherlerIlke Anac Sakir

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