Purpose: To demonstrate the use of viability rt-PCR to discriminate between living pathogenic Listeria and non-viable Listeria phage/ Listeria DNA in a cheese production setting.
Methods: Mozzarella cheese was obtained from a cheese manufacturer. To test for effect of PMA treatment on the detection of Listeria spp. in cheese treated with Listeria phage, cultures were grown in Listeria ONE Broth, 26 h at 30°C. QIAGEN QIAsymphony workflows were applied. Two studies were performed: One assessed the detection of Listeria spp. on uninoculated cheese cultures with and without PMA treatment. The second was performed with low level L. monocytogenes inoculation, with and without PMA treatment.
Results: In the uninoculated study, Listeria spp. was detected in 4 cultures. PMA treatment completely masked the Listeria spp. signal. This suggests that there was dead Listeria DNA present in these cheese cultures, most likely derived from the Listeria phage solution applied to the cheese during manufacture. In the spiking study, Listeria spp. was detected in 19 culture replicates. PMA treatment completely masked the rt-PCR signal in 6 of these and resulted in significant Ct shifts toward higher values in the other 12. PMA (viability rt-PCR) masks the signal from the Listeria phage DNA but does not decrease the detection rate of living Listeria monocytogenes cells.
Significance: Viability rt-PCR can be used to increase the power of rt-PCR in live-dead differentiation of pathogens in a number of settings of interest to the food industry.