| 1. |
- Fachmann, M S R, et al.
(författare)
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Detection of Salmonella in meat in less than 5 hours by a low-cost and non-complex sample preparation method.
- 2017
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 83:5
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Tidskriftsartikel (refereegranskat)abstract
- Salmonella is recognised as one of the most important foodborne bacteria, and has a wide health and socioeconomical impact worldwide. Fresh pork meat is one of the main sources of Salmonella and efficient and fast methods for detection are therefore necessary. Current methods for Salmonella detection in fresh meat usually include >16 h of culture enrichment, in few cases < 12 h, thus requiring at least two working shifts. Here, we report a rapid (< 5 h) and high throughput method, for screening of Salmonella in samples from fresh pork meat, consisting of a 3-h enrichment in standard buffered peptone water, and a real-time PCR compatible sample preparation method, based on filtration, centrifugation, and enzymatic digestion, followed by fast cycling real-time PCR detection. The method was validated in an un-paired, comparative study against the Nordic Committee on Food Analysis (NMKL) reference culture method 187. Pork meat samples (n=140) were either artificially contaminated with Salmonella at levels: 0, 1-10 and 10-100 CFU/25 g, or naturally contaminated. Cohen's Kappa for degree of agreement between the rapid method and the reference was 0.64 and the relative accuracy, sensitivity and specificity for the rapid method were 81.4, 95.1 and 97.9 %, respectively. The limit of detection (LOD50) was 8.8 CFU/25 g for the rapid method and 7.7 CFU/25 g for the reference method. Implementation of this method will enable faster release of Salmonella low risk meat, providing savings for meat producers, and help contribute to improved food safety.IMPORTANCE: While the cost of analysis and hands-on time of the presented rapid method were comparable to reference culture methods, the fast product release by this method can provide the meat industry with a competitive advantage. Not only will the abattoirs save costs for work hours and cold storage; consumers as well as retailers will also benefit from fresher meat with a longer shelf life. Furthermore, the presented sample preparation might be adjusted for application in detection of other pathogenic bacteria in different sample types.
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| 2. |
- Grønlund, H. A., et al.
(författare)
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The use of infrared thermography as a novel approach for real-time validation of PCR thermocyclers
- 2010
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Ingår i: Food Analytical Methods. - : Springer Science and Business Media LLC. - 1936-9751 .- 1936-976X. ; 3:2, s. 116-119
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Tidskriftsartikel (refereegranskat)abstract
- Validation of PCR thermocycler performance is crucial to obtain reliable results. In this study, infrared (IR) thermography was evaluated as a novel validation tool. After stabilisation, no significant difference in the temperatures recorded using thermography and a reference block-based system was found. By employing IR thermography, information about the length of the time until temperature stabilisation in the sample could be obtained. This study shows the potential of using IR thermography for validation of thermocyclers. © Springer Science + Business Media, LLC 2009.
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| 3. |
- Harder, C. B., et al.
(författare)
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Molecular diagnostics of salmonella and campylobacter in human/animal fecal samples remain feasible after long-term sample storage without specific requirements
- 2021
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Ingår i: AIMS Microbiology. - 2471-1888. ; 7:4, s. 399-414
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Tidskriftsartikel (refereegranskat)abstract
- Rapid advances in the development of sequencing technologies, numbers of commercial providers and diminishing costs have made DNA-based identification and diagnostics increasingly accessible to doctors and laboratories, eliminating the need for local investments in expensive technology and training or hiring of skilled technicians. However, reliable and comparable molecular analyses of bacteria in stool samples are dependent on storage and workflow conditions that do not introduce post-sampling bias, the most important factor being the need to keep the DNA at a stable detectable level. For that reason, there may remain other prohibitively costly requirements for cooling or freezing equipment or special chemical additives. This study investigates the diagnostic detectability of Salmonella and Campylobacter DNA in human, pig and chicken stool samples, stored at different temperatures and with different preservation methods. Stool samples were spiked with 106 CFU/mL of both Salmonella and Campylobacter strains stored at −20 ℃, 5 ℃ and 20 ℃ (Room temperature, RT) and treated with either RNAlater, EDTA or Silica/ethanol. DNA was extracted at 9 different time points within 30 days and quantified by Qubit (total DNA) and qPCR (Salmonella and Campylobacter DNA). We found no statistically significant differences among the different preservation methods, and DNA from both species was easily detected at all time points and at all temperatures, both with and without preservation. This suggests that infections by these bacteria can be diagnosed and possibly also analysed in further detail simply by taking a stool sample in any suitable sealed container that can be transported to laboratory analysis without special storage or preservation requirements. We briefly discuss how this finding can benefit infection control in both developed and developing countries.
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| 4. |
- Jakočiune, D., et al.
(författare)
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Enumeration of salmonellae in table eggs, pasteurized egg products, and egg-containing dishes by using quantitative real-time PCR
- 2014
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 80:5, s. 1616-1622
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Tidskriftsartikel (refereegranskat)abstract
- Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (qPCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes. Salmonella enterica serovar Enteritidis and S. enterica serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between the numbers of salmonellae and the quantification cycle (Cq) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate of salmonellae in two egg-containing dishes, long egg and tiramisu, at abused refrigeration temperatures, and results indicated the growth of bacteria over a 1-week period. In conclusion, enrichment qPCR was shown to be reliable for enumeration of salmonellae in different egg products. © 2014, American Society for Microbiology.
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| 5. |
- Josefsen, M. H., et al.
(författare)
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Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time pcr and propidium monoazide treatment, as a tool for quantitative risk assessment
- 2010
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 76:15, s. 5097-5104
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Tidskriftsartikel (refereegranskat)abstract
- A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (CT.) values (R2 = 0.993), with a quantification range of 1 × 102 to 1 × 107 CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R 2 = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment. Copyright © 2010, American Society tor Microbiology. All Rights Reserved.
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| 6. |
- Søndergaard, M. S. R., et al.
(författare)
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Low-cost monitoring of Campylobacter in poultry houses by air sampling and quantitative PCR
- 2014
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Ingår i: Journal of Food Protection. - 0362-028X .- 1944-9097. ; 77:2, s. 325-330
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Tidskriftsartikel (refereegranskat)abstract
- The present study describes the evaluation of a method for the quantification of Campylobacter by air sampling in poultry houses. Sampling was carried out in conventional chicken houses in Poland, in addition to a preliminary sampling in Denmark. Each measurement consisted of three air samples, two standard boot swab fecal samples, and one airborne particle count. Sampling was conducted over an 8-week period in three flocks, assessing the presence and levels of Campylobacter in boot swabs and air samples using quantitative real-time PCR. The detection limit for air sampling was approximately 100 Campylobacter cell equivalents (CCE)/m3. Airborne particle counts were used to analyze the size distribution of airborne particles (0.3 to 10 mm) in the chicken houses in relation to the level of airborne Campylobacter. No correlation was found. Using air sampling, Campylobacter was detected in the flocks right away, while boot swab samples were positive after 2 weeks. All samples collected were positive for Campylobacter from week 2 through the rest of the rearing period for both sampling techniques, although levels 1- to 2-log CCE higher were found with air sampling. At week 8, the levels were approximately 104 and 105 CCE per sample for boot swabs and air, respectively. In conclusion, using air samples combined with quantitative realtime PCR, Campylobacter contamination could be detected earlier than by boot swabs and was found to be a more convenient technique for monitoring and/or to obtain enumeration data useful for quantitative risk assessment of Campylobacter. © International Association for Food Protection.
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| 7. |
- 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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| 8. |
- Hansen, T., et al.
(författare)
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Evaluation of Direct 16S rDNA sequencing as a metagenomics-based approach to screening bacteria in bottled water
- 2013
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Ingår i: Biosecurity and bioterrorism. - : Mary Ann Liebert Inc. - 1538-7135 .- 1557-850X. ; 11:SUPPL. 1, s. S158-S165
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Tidskriftsartikel (refereegranskat)abstract
- Deliberate or accidental contamination of food, feed, and water supplies poses a threat to human health worldwide. A rapid and sensitive detection technique that could replace the current labor-intensive and time-consuming culture-based methods is highly desirable. In addition to species-specific assays, such as PCR, there is a need for generic methods to screen for unknown pathogenic microorganisms in samples. This work presents a metagenomics-based direct-sequencing approach for detecting unknown microorganisms, using Bacillus cereus (as a model organism for B. anthracis) in bottled water as an example. Total DNA extraction and 16S rDNA gene sequencing were used in combination with principle component analysis and multicurve resolution to study detection level and possibility for identification. Results showed a detection level of 10 5 to 106 CFU/L. Using this method, it was possible to separate 2 B. cereus strains by the principal component plot, despite the close sequence resemblance. A linear correlation between the artificial contamination level and the relative amount of the Bacillus artificial contaminant in the metagenome was observed, and a relative amount value above 0.5 confirmed the presence of Bacillus. The analysis also revealed that background flora in the bottled water varied between the different water types that were included in the study. This method has the potential to be adapted to other biological matrices and bacterial pathogens for fast screening of unknown bacterial threats in outbreak situations. © 2013, Mary Ann Liebert, Inc.
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| 9. |
- Josefsen, M. H., et al.
(författare)
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Diagnostic PCR : Comparative sensitivity of four probe chemistries
- 2009
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Ingår i: Molecular and Cellular Probes. - : Elsevier BV. - 0890-8508 .- 1096-1194. ; 23:3-4, s. 201-203
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Tidskriftsartikel (refereegranskat)abstract
- Three probe chemistries: locked nucleic acid (LNA), minor groove binder (MGB) and Scorpion were compared with a TaqMan probe in a validated real-time PCR assay for detection of food-borne thermotolerant Campylobacter. The LNA probe produced significantly lower Ct-values and a higher proportion of positive PCR responses analyzing less than 150 DNA copies than the TaqMan probe. Choice of probe chemistry clearly has an impact on the sensitivity of PCR assays, and should be considered in an optimization strategy.
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| 10. |
- Knutsson, Rickard, et al.
(författare)
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Modeling of 5′ nuclease real-time responses for optimization of a high-throughput enrichment PCR procedure for Salmonella enterica
- 2002
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 40:1, s. 52-60
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Tidskriftsartikel (refereegranskat)abstract
- The performance of a 5′ nuclease real-time PCR assay was studied to optimize an automated method of detection of preenriched Salmonella enterica cells in buffered peptone water (BPW). The concentrations and interactions of the PCR reagents were evaluated on the basis of two detection responses, the threshold cycle (Cτ) and the fluorescence intensity by a normalized reporter value (ΔRn). The Cτ response was identified as the most suitable for detection modeling to describe the PCR performances of different samples. DNA extracted from S. enterica serovar Enteritidis was studied in double-distilled H2O (ddH2O) and in two different enrichment media (brain heart infusion and BPW) with two PCR mixtures based on AmpliTaq Gold or rTth. A descriptive model was proposed and fitted to the available experimental data. Equivalent PCR performances for the two PCR mixtures were obtained when DNA was diluted in ddH2O. However, the level of detection of DNA was affected when BPW was present during amplification. Use of the rTth mixture generated a 1-log-unit wider linear range of amplification, and the DNA detection levels were 2 × 10-12 g/microwell for the rTth mixture and 2 × 10-12 g/microwell for the AmpliTaq Gold mixture. To verify the improved amplification capacity of the rTth mixture, BPW was inoculated with 1 CFU of S. enterica serovar Enteritidis per ml and the mixture was incubated at 30°C. Samples for PCR were withdrawn every 4 h during a 36-h enrichment. Use of the rTth mixture resulted in an earlier PCR detection during enrichment than use of the AmpliTaq Gold mixture. For accurate detection (Cτ ≤ 30) of S. enterica serovar Enteritidis inoculated in BPW, the rTth mixture required 8.4 h of enrichment, while the AmpliTaq Gold mixture needed 11.6 h. In conclusion, the principle applied can improve the methodology of 5′ nuclease real-time PCR for numerical optimization of sample pretreatment strategies to provide automated diagnostic PCR procedures.
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