Pre-PCR treatments as a key factor on the probability of detection of Listeria monocytogenes and Salmonella in ready-to-eat meat products by real-time PCR

The aim of this study was to compare the performance of different pre-PCR treatments for the detection of Listeria monocytogenes and Salmonella spp. by real-time PCR in spiked ready-to-eat meat samples (cooked ham, dry-cured ham and fermented sausage). Three different pre-PCR treatments were assayed: (i) PrepMan® Ultra Sample Preparation Reagent, (ii) DNA purification using DNeasy Tissue Kit and QIAcube automated sample preparation system, and (iii) BAX® system. The analysis range was 2 to 2 × 106 CFU/ml. PCR was done in quintuplicate and the experiment was repeated five times. The ‘probability of detection’ was used to compare the efficiency of the pre-PCR treatments and to calculate the theoretical limit of detection of each treatment. The best pre-PCR treatment was DNA purification using DNeasy Tissue kit and QIAcube; it showed the highest detection probability and the lowest limit of detection for each food type and pathogen assayed, followed by PrepMan and BAX treatments. Probability of detection showed differences among food matrices and between L. monocytogenes and Salmonella detection probability. Despite being an expensive method, DNeasy Tissue kit would be the recommended treatment to reduce the risk of false negative results and to improve detection of foodborne pathogens in RTE-meat products by real-time PCR.