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

摘要

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 50g/ml, 20min incubation time, and 30min light exposure time were suggested to use for detecting viable cells. When the inoculum concentration was 10(3) CFU/100ml, 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 applicationsTo 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 10(3) CFU/100ml. 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.