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- Grønlund, H., et al.
(författare)
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Microarray-based genotyping of Salmonella : Inter-laboratory evaluation of reproducibility and standardization potential
- 2011
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Ingår i: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605 .- 1879-3460. ; 145:SUPPL. 1, s. S79-S85
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial food-borne infections in humans caused by Salmonella spp. are considered a crucial food safety issue. Therefore, it is important for the risk assessments of Salmonella to consider the genomic variation among different isolates in order to control pathogen-induced infections. Microarray technology is a promising diagnostic tool that provides genomic information on many genes simultaneously. However, standardization of DNA microarray analysis is needed before it can be used as a routine method for characterizing Salmonella isolates across borders and laboratories. A comparative study was designed in which the agreement of data from a DNA microarray assay used for typing Salmonella spp. between two different labs was assessed. The study was expected to reveal the possibility of obtaining the same results in different labs using different equipment in order to evaluate the reproducibility of the microarray technique as a first step towards standardization. The low-density array contains 281 57-60-mer oligonucleotide probes for detecting a wide range of specific genomic marker genes associated with antibiotic resistance, cell envelope structures, mobile genetic elements and pathogenicity. Several critical methodology parameters that differed between the two labs were identified. These related to printing facilities, choice of hybridization buffer, wash buffers used following the hybridization and choice of procedure for purifying genomic DNA. Critical parameters were randomized in a four-factorial experiment and statistical measures of inter-lab consistency and agreement were performed based on the kappa coefficient. A high level of agreement (kappa = 0.7-1.0) in microarray results was obtained even when employing different printing and hybridization facilities, different procedures for purifying genomic DNA and different wash buffers. However, less agreement (Kappa = 0.2-0.6) between microarray results were observed when using different hybridization buffers, indicating this parameter as being highly critical when transferring a standard microarray assay between laboratories. In conclusion, this study indicates that DNA microarray assays can be reproduced in at least two different facilities, which is a pre-requisite for the development of standard guidelines. © 2010 Elsevier B.V.
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| 2. |
- Krämer, N., et al.
(författare)
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A novel strategy to obtain quantitative data for modelling : Combined enrichment and real-time PCR for enumeration of salmonellae from pig carcasses
- 2011
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Ingår i: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605 .- 1879-3460. ; 145:SUPPL. 1, s. S86-S95
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Tidskriftsartikel (refereegranskat)abstract
- Salmonella is a major zoonotic pathogen which causes outbreaks and sporadic cases of gastroenteritis in humans worldwide. The primary sources for Salmonella are food-producing animals such as pigs and poultry. For risk assessment and hazard analysis and critical control point (HACCP) concepts, it is essential to produce large amounts of quantitative data, which is currently not achievable with the standard cultural based methods for enumeration of Salmonella. This study presents the development of a novel strategy to enumerate low numbers of Salmonella in cork borer samples taken from pig carcasses as a first concept and proof of principle for a new sensitive and rapid quantification method based on combined enrichment and real-time PCR. The novelty of the approach is in the short pre-enrichment step, where for most bacteria, growth is in the log phase. The method consists of an 8h pre-enrichment of the cork borer sample diluted 1:10 in non-selective buffered peptone water, followed by DNA extraction, and Salmonella detection and quantification by real-time PCR. The limit of quantification was 1.4 colony forming units (CFU)/20cm2 (approximately 10g) of artificially contaminated sample with 95% confidence interval of±0.7 log CFU/sample. The precision was similar to the standard reference most probable number (MPN) method. A screening of 200 potentially naturally contaminated cork borer samples obtained over seven weeks in a slaughterhouse resulted in 25 Salmonella-positive samples. The analysis of salmonellae within these samples showed that the PCR method had a higher sensitivity for samples with a low contamination level (<6.7CFU/sample), where 15 of the samples negative with the MPN method was detected with the PCR method and 5 were found to be negative by both methods. For the samples with a higher contamination level (6.7-310CFU/sample) a good agreement between the results obtained with the PCR and MPN methods was obtained. The quantitative real-time PCR method can easily be applied to other food and environmental matrices by adaptation of the pre-enrichment time and media. © 2010 Elsevier B.V.
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| 3. |
- Löfström, Charlotta, et al.
(författare)
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Culture-independent quantification of Salmonella enterica in carcass gauze swabs by flotation prior to real-time PCR
- 2011
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Ingår i: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605 .- 1879-3460. ; 145:SUPPL. 1, s. S103-S109
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Tidskriftsartikel (refereegranskat)abstract
- To facilitate quantitative risk assessment in the meat production chain, there is a need for culture-independent quantification methods. The aim of this study was to evaluate the use of flotation, a non-destructive sample preparation method based on traditional buoyant density centrifugation, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100cm2) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant density of two Salmonella strains in different growth conditions was determined to be 1.065-1.092g/ml. Based on these data, an optimal discontinuous flotation with three different density layers, ~1.200, 1.102 and 1.055g/ml, was designed for extracting intact Salmonella cells from pig carcass swabs. The method allowed accurate quantification from 4.4×102 to at least 2.2×107CFU Salmonella per swab sample using qPCR (without preceding DNA extraction) or selective plating on xylose lysine deoxycholate agar. Samples with 50CFU could be detected occasionally but fell outside the linear range of the standard curve. The swab samples showed a broad biological diversity; for seven samples not inoculated with Salmonella, the microbial background flora (BGF) was determined to 5.0±2.2 log CFU/ml sample withdrawn after flotation. It was determined that the proceeding PCR step was inhibited by BGF concentrations of ≥6.1×108CFU/swab sample, but not by concentrations ≤6.1×106CFU/swab sample. By using the gauze swabs directly in the flotation procedure, the homogenization step normally used for preparation of food-related samples could be excluded, which simplified the culture-independent quantification method considerably. © 2010 Elsevier B.V.
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