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Rapid determination of viable but non-culturable Escherichia coli O157:H7 and Salmonella enterica in fresh produce by loop-mediated isothermal amplification coupling propidium monoazide treatment Han, Lu


Escherichia coli O157:H7 and Salmonella enterica are reported to be the leading causes of foodborne outbreaks linked to fresh produce. Both bacteria can enter “viable but non-culturable (VBNC)” state that preclude detection by using the conventional cultural-based methods. In this study, we developed a method of combining propidium monoazide (PMA) and loop-mediated isothermal amplification (LAMP) to determine VBNC E. coli O157:H7 and S. enterica in fresh produce. PMA-LAMP assays targeting wzy gene of E. coli O157:H7 and agfA gene of S. enterica were evaluated in pure culture and spiked tomato, lettuce and spinach, and compared with PMA-qPCR. No cross-reaction was observed in the specificity tests. The limit of detection of PMA-LAMP was 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture, and 5.1310³⁻⁴ CFU/g for VBNC E. coli O157:H7 and 1.05 10⁴⁻⁵ CFU/g for VBNC S. enterica in fresh produce. Standard curves had the correlation coefficient ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for both VBNC microbes. Compared to PMA-qPCR, PMA-LAMP saved substantial amount of time (i.e. 20 min vs. 1 h) to determine enteric bacterial pathogens while achieved comparable sensitivity and quantitative capacity. Taken together, PMA-LAMP is a rapid, sensitive and robust method to detect and quantify VBNC E. coli O157:H7 and S. enterica in fresh produce, and has the potential for food microbiological surveillance in resource-limited settings.

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