The sample preparation in the PCR method consists of non-selective enrichment in BPW followed by centrifugation and automated DNA extraction. The use of automated DNA extraction in combination with the closed system of real-time PCR provides a fast and less laborious method with minimized risk of contamination. Furthermore, the real-time PCR method can easily be adapted to include the dUTP-uracil-N-glycosylase (UNG) system, minimizing the risk of carryover contamination
. The PCR reagents used in the method can be mixed in advance, distributed in smaller, ready-to-use quantities, and frozen at GDC-0941 molecular weight -20°C for up to 3 months . These features are a major benefit for on-site use of the test at the slaughterhouses. The method is an open-formula technique, i.e., the reagents and target gene, etc., are known, in contrast to commercial kits. However, further decreasing the total time for analysis to below 8 h will certainly be even more beneficial to industry and is a challenge in the further developing of the method. The prevalence of Salmonella in Danish pork meat and broiler flocks is low (0.9% and 2.2%, respectively ). Therefore, samples artificially contaminated with Salmonella in the exponential growth phase stressed by a cold storage
overnight to simulate the condition under production of poultry and pork meat were used for the majority of the samples included in the validation study. This alternative to naturally contaminated samples is in compliance with international
guidelines buy LY3023414 [15, 19]. However, naturally MG-132 manufacturer contaminated swab samples were used for the comparative trial. The NMKL-71 (1999) method  was chosen as the reference method because it is used in the Nordic countries instead of the ISO 6579:2002 method . The difference in the two methods is that in the NMKL method only one selective enrichment media is used Rappaport Vassiliades soy broth (RVS) instead of two in the ISO method (RVS and Muller-Kauffmann Tetrathionate-Novobiocin broth, MKTTn). The methods have been determined to be equal to the respective part of the ISO method . The real-time PCR method amplifies a part of the ttrRSBCA locus used for tetrathionate respiration in Salmonella. The relative selectivity of the PCR assay (primers and probes) has previously been found to be 100% when tested on 110 Salmonella strains and 87 find more non-Salmonella strains . Therefore, this parameter was excluded from the comparative test performed in this study, in accordance with NordVal guidelines. The relative accuracy, sensitivity and specificity were evaluated for the PCR method in comparison with the standard culture-based method currently in use for detection of Salmonella  according to the NordVal protocol (Table 1). Two of the artificially contaminated poultry neck-skins were found positive by the real-time PCR method and negative by the reference method.