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We developed a 12-h Salmonella detection method, based on 8 h of preenrichment, followed by automated DNA extraction and a sensitive real-time PCR. The method was optimized to obtain the highest possible yield of cells and DNA. The growth of different Salmonella strains in various preenrichment media and the effects of adding growth-promoting and selective reagents were explored, taking into account their PCR compatibility. The effects of (i) analyzing larger volumes (1 to 5 ml) from preenriched samples and introducing wash steps prior to DNA extraction, (ii) regulating the amount of paramagnetic particles (increasing it from 60 to 90 l) in the DNA extraction, (iii) eluting the DNA in reduced volumes (25 or 50 l rather than 100 l), and (iv) increasing the PCR template volume (from 5 to 20 l) were investigated. After 8 h of preenrichment, buffered peptone water yielded the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 l were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 l) had no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 l improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples, with a relative accuracy of 99%, a relative sensitivity of 98%, and a relative specificity of 100%.

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As part of a large international project for standardization of PCR (Food-PCR; www.pcr.dk), a multiplex, multiplatform, ready-to-go enrichment followed by a real-time PCR method, including an internal amplification control, was developed for detection of food-borne thermotolerant campylobacters in chickens. Chicken rinse samples were enriched in Bolton broth for 20 h, a simple and rapid (1-h) resin-based DNA extraction was performed, and DNA samples were then tested with two instrument platforms: ABI-PRISM 7700 and RotorGene 3000. The method was validated against an International Standard Organization (ISO)-based culture method by testing low, medium, and high levels of 12 spiked and 66 unspiked, presumably naturally contaminated, chicken rinse samples. In the RotorGene, a positive PCR response was detected in 40 samples of the 66. This was in complete agreement with the enriched ISO culture. The ABI-PRISM 7700 missed one culture-positive sample. Positive samples contained 10 2 to 10 7 CFU/ml after enrichment in Bolton broth. In the enriched samples a detection probability of 95% was obtained at levels of 1 ؋ 10 3 and 2 ؋ 10 3 CFU/ml in the RotorGene and ABI-PRISM, respectively. The amplification efficiency in both platforms was 90%, although the linear range of amplification of purified genomic DNA was 1.5 ؋ 10 1 to 1 ؋ 10 7 (R 2 ‫؍‬ 1.00) for the RotorGene and

To identify a cost-effective and practical method for detection of methicillin-resistant Staphylococcus aureus (MRSA) in pig herds, the relative sensitivity of four sample types: nasal swabs, ear-skin (skin behind the ears) swabs, environmental dust swabs and air was compared. Moreover, dependency of sensitivity on within-herd prevalence was estimated. spa-typing was applied in order to study strain diversity. The sensitivity of one air sample was equal to the sensitivity of ten pools of five nasal swabs and relatively independent of within-herd prevalence [predicted to be nearly perfect (99%) for within-herd prevalence ⩾25%]. The results indicate that taking swabs of skin behind the ears (ten pools of five) was even more sensitive than taking nasal swabs (ten pools of five) at the herd level and detected significantly more positive samples. spa types t011, t034 and t4208 were observed. In conclusion, MRSA detection by air sampling is easy to perform, reduces costs and analytical time compared to existing methods, and is recommended for initial testing of herds. Ear-skin swab sampling may be more sensitive for MRSA detection than air sampling or nasal swab sampling.