Detection of Live Listeria monocytogenes by EMA-qPCR

Abstract:

An ethidium monoazide in combination with real-time PCR method (EMA-PCR) was newly developed for selective detection of live Listeria monocytogenes cells from dead cells. The primers and TaqMan probes were designed using the hly gene which encoded listeriolysin O (LLO). The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of Listeria monocytogenes strains and other pathogenic bacteria strains (n=56). A qPCR assay was detected with EMA of different concentrations. Sodium deoxycholate solution (SD) was used to strengthen the inhibitory effect. In addition, 73 various artificial contaminated food and environmental samples (braised chicken, milk, minced meat and garbage leachate) were investigated for L. monocytogenes. The primer probe accurately detected L. monocytogenes without specific amplification for other strains. EMA could penetrate dead cells resulting in the covalent links of DNA upon 15 min light exposure and therefore inhibition of amplification under an optimal concentration of 2.5 μg/mL with a detection limit of 150 CFU/mL. The Ct value of live Lm increased under SD treatment. The diagnostic accuracy of EMA-qPCR method was up to 100 % compared to the traditional culture method. This method with simple operation, time-saving and high efficiency has a promising application on accurate microbiological monitoring of food safety and environmental source.