Rapid detection of Salmonella in meat: Comparative and collaborative validation of a non-complex and cost effective pre-PCR protocol

Cost-effective and rapid monitoring of Salmonella in the meat production chain can contribute to food safety. The objective was, for the first time, to validate an easy-to-use pre-PCR sample preparation method based on a simple boiling protocol for screening of Salmonella in meat and carcass swab samples using a real-time PCR method. The protocol included incubation in buffered peptone water, centrifugation of an aliquot and a boiling procedure. The validation study included comparative and collaborative trials recommended by the Nordic Organization for Validation of Alternative Methods (NordVal). The comparative trial was performed against a culture based reference method (NMKL187, 2007) and a previously NordVal approved PCR method with a semi-automated magnetic bead-based DNA extraction step using 122 artificially contaminated samples. The limit of detection (LOD50) was found to be 3.0, 3.2 and 3.4 CFU/sample for the boiling, magnetic bead-based and NMKL187 methods, respectively. When comparing the boiling method with the magnetic beads, the relative accuracy (AC), relative sensitivity (SE) and relative specificity (SP) were found to be 98%, 102% and 98%, respectively (Cohen’s kappa index 0.95). When comparing results obtained by the boiling to the culture based method, the AC, SE and SP were found to be 98%, 102% and 98%, respectively (kappa index 0.93). In the collaborative trial valid results from 11 laboratories were included and apart from two false positive samples, no deviating results were obtained (SP, SE and AC were 100%, 95% and 97%, respectively). This test is under implementation by the Danish meat industry, and can be useful for screening of large number of samples in the meat production, especially for fast release of minced meat with short shelf-life.