Rapid Determination of Viable but Non-culturable Escherichia coli O157:H7 and Salmonella enterica in Fresh Produce by Loop-mediated Isothermal Amplification Coupled with a Propidium Monoazide Treatment.

In this study, we assessed propidium monoazide (PMA)-qPCR assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting wzy gene of E. coli O157:H7 and agfA gene of S. enterica and PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce and spinach. No cross-reaction was observed in the specificity tests. The limit of detection (LOD) with PMA-LAMP was 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture, and 5.13×103-4 CFU/g for VBNC E. coli O157:H7 and 1.05×104-5 CFU/g for VBNC S. enterica in fresh produce, which was comparable to that of PMA-qPCR. Standard curves showed a correlation coefficient ranging from 0.925 to 0.996, indicating good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min vs. 1 h) and achieved comparable sensitivity and quantitative capacity to a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce.IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade the detection in the conventional plating assays. ...

Source: Applied and Environmental Microbiology - January 30, 2020 Category: Microbiology Authors: Han L, Wang K, Ma L, Delaquis P, Bach S, Feng J, Lu X Tags: Appl Environ Microbiol Source Type: research