Quantitative detection of viable Escherichia coli O157:H7 using a photoreactive DNA-binding dye propidium monoazide in irrigation water.

• LSCM and microplate reader were used to verify the effect of PMA treatment. • Addition of dead bacteria (˜104 CFU/mL) did not affect detection of viable bacteria. • Low concentration of viable bacteria (102 CFU/mL) is detectable in irrigation water. • The method is sensitive and accurate for bacteria detection in irrigation water. Escherichia coli O157:H7 is a kind of waterborne pathogen and recognized as a common pathogenic microorganism in irrigation water. In this study, a quantitative PCR (qPCR) assay with propidium monoazide (PMA) treatment called PMA-qPCR assay was developed to detect the presence of E. coli O157:H7 in natural irrigation water that was more specific, more sensitive, and was unaffected by dead cells compared with traditional and conventional methods. Specific gene, fliC from E. coli O157:H7 was selected to design the unique primer and probe for the assay while propidium monoazide (PMA) was used to effectively prevent the interference of DNA from dead cells. Evaluation of the effect of PMA treatment exhibited permeation of the dead cell membranes that was observed under confocal laser scanning microscope (CLSM). The PMA-qPCR assay efficiently detected viable cells even in the presence of 104 CFU/mL dead cells. Under optimized conditions the limit of detection for viable cells was 102 CFU/mL or 10 CFU/mL in pure culture and spiked irrigation water in a 3 h assay. The PMA-qPCR assay did not show significant difference in the absence of the dead bacteria indicating specificity and selectivity for viable E. coli O157:H7. The PMA-qPCR assay developed in this study has potential for precise and sensitive detection of viable E. coli O157:H7 in irrigation water. [ABSTRACT FROM AUTHOR]