Detection of Viable Escherichia coli O157:H7 in Apple Juice and Spinach Wash Water Using a Concentrating Pipette and Ethidium Monoazide-real-time PCR

Introduction: Escherichia coli O157:H7 associated with food has caused many serious public health problems in recent years.  Two significant limitations to the use of more sensitive and selective microbiological detection methods in the food industry include: (a) the need for a prolonged sample enrichment prior to analysis, and (b) the inability to differentiate between viable and dead cells. 

Purpose: The objective of this study was to develop and optimize a method that combines a novel cell concentration step with real-time (RT)-PCR to detect only viable E. coli O157:H7 cells rapidly without the need for sample enrichment. 

Methods: Apple juice and spinach wash water were artificially contaminated by different concentrations of viable and dead E. coli O157:H7 cells and concentrated using a Concentrating Pipette (CP) designed by InnovaPrep LLC.  Samples were further purified by immunomagnetic separation and treated with ethidium monoazide (EMA), a dye that can penetrate dead cells and bind to cellular DNA.  DNA was extracted and amplified by TaqMan® RT-PCR targeting the uidA gene to detect only viable E. coli O157:H7 cells.

Results: This assay could detect as low as 3 CFU ml^-1 of viable E. coli O157:H7 in apple juice and 3000 CFU ml^-1 in spinach wash water within 4 h.  In addition, it completely prevented false-positive PCR results generated by 10³ CFU ml^-1 and 10 CFU ml^-1 of dead E. coli O157:H7 cells in apple juice and spinach wash water, respectively.

Significance: In conclusion, the CP-EMA-RT-PCR assay can effectively detect viable E. coli O157:H7 cells in apple juice and spinach wash water within a short time without the need for sample enrichment, while preventing the amplification of DNA in dead cells.