Purpose: The purpose of this study was to evaluate an enzymatic approach to remove free Listeria DNA present in food samples treated with commercial phage suspension without affecting the PCR detection of living Listeriacells.
Methods: Dilutions of living L. monocytogenes bacteria were spiked into 25g of Cheddar cheese portions that were treated or not with Listeria phage suspension. Samples were enriched for 24h in 225 mL of Listeria Special Broth. 100 µL aliquots were submitted to an enzymatic treatment and Listeria DNA was subsequently extracted with a specific lysis buffer and PCR amplified with the iQ-Check® Listeria spp. assay. Cq values for both Listeria targets and Internal Control were compared in presence or absence of the free DNA removal treatment.
Results: Treating Cheddar cheese with Listeria phage led to a positive PCR signal with Cq in the 35-37 range, corresponding to approximately 5x104 bacterial genomes per gram of cheese. This signal hindered the detection of living bacteria. When the sample was pretreated with the enzyme mix, free DNA was readily suppressed whereas living cells were detectable even at the lowest amount. Furthermore, the Internal Control signal was not impacted confirming that the extraction step efficiently neutralized the enzymatic reagent.
Significance: These results demonstrate that a straightforward enzymatic method is efficient to clean food samples treated with phage suspension from free DNA without affecting the detection of living cells. The use of this method can be easily extended to other food matrices that are prone to the presence of dead cells.