Molecular monitoring of disinfection efficacy of E. coli O157:H7 in bottled purified drinking water by quantitative PCR with a novel dye

Abstract

A new method was developed using a novel dye thiazole orange monoazide (TOMA) combined with quantitative real‐time PCR (qPCR) to detect viable Escherichia coli O157:H7 cells. Different from the commonly used PMA‐qPCR assay that is based on membrane integrity, this TOMA‐qPCR method is based on the concept of metabolic activity. TOMA consists of three components: a nucleic acid‐intercalating moiety, a crosslinkable moiety and a linker. TOMA concentration at 50 μg/ml, 20 min incubation time, and 30 min light exposure time were suggested to use for detecting viable cells. When the inoculum concentration was 103 CFU/100 ml, TOMA‐qPCR assay could completely exclude the effect of dead cells treated with heat, chlorine, or UV. Moreover, TOMA‐qPCR could also be used to detect viable but nonculturable state (VBNC) cells. The result shows that TOMA‐qPCR would be an alternative choice for the detection of viable cells. Practical applications To specifically detect only viable cells is of great importance in most detection of microbial diagnostics, especially for detection of foodborne pathogens such as E. coli O157:H7, which could cause health risk at low concentrations. The existing detection methods have some defects in detecting only viable cells. In this article, a novel dye was developed and combined with qPCR, to monitor sanitizing efficacy of different disinfection methods on E. coli O157:H7 at low concentrations. When detecting viable E. coli in bottled water, the established TOMA‐qPCR assay completely exclude the effect of dead cells when the inoculated concentration is 103 CFU/100 ml. This method is also suitable for the detection of VBNC samples. Therefore, this method can be used to detect pathogenic bacteria with low contamination under extreme conditions such as low temperature, oligotrophic, strong radiation, and so on.

Detection and Quantification Methods for Viable but Non-culturable (VBNC) Cells in Process Wash Water of Fresh-Cut Produce: Industrial Validation

“…However, the PCR signal of L. monocytogenes DNA activated with PMAxx (75 µM) obtained from chorinetreated cells was not completely inactivated, showing a Ct value below the LOD (Figure 3). Our results agree with a previous study that observed that PMA-qPCR assay (PMA at 50 µM for 20 min of incubation at 37 • C) did not reduce the signal of chlorine-killed cells of E. coli O:157 H7 (10 4 cfu/ml) artificially inoculated in drinking water (Cao et al, 2019a). Based on these results, new experiments were performed under different conditions of time/temperature for the incubation of the DNA with the dye.…”

Recovery of Mycobacteria from Heavily Contaminated Environmental Matrices

“…In viable cells, with an intact envelope, the v-qPCR dye cannot penetrate inside and thus its DNA is free for amplification in a subsequent qPCR assay. A number of v-qPCR dyes have been developed for viability studies: EMA (ethidium monoazide), PMA (propidium monoazide) [ 45 ], and TOMA (thiazole orange monoazide) [ 46 ]. The utility of the PMA v-qPCR method has been demonstrated for evaluating the efficiency of water disinfection, sensitivity of M. avium ssp.…”

Recent trends for detection, quantification and identification of microorganisms in food industry

“…More recently, the "viability-PCR" approach has been proposed as an alternative method to RT-qPCR, based on the treatment of the sample with DNA intercalating dye, ethidium monoazide (EMA) or propidium monoazide (PMA) or thiazole orange monoazide (TOMA) which penetrates only into cells with damaged membranes or binds to free DNA, inhibiting subsequent amplification. These results related to the amplification show that living cells with intact membranes are viable (Cao et al, 2018;Gobert et al, 2018;Scariot et al, 2018).…”