| 1. |
- Hoorfar, Jeffrey, et al.
(författare)
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Diagnostic PCR: validation and sample preparation are two sides of the same coin
- 2004
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Ingår i: APMIS : acta pathologica, microbiologica, et immunologica Scandinavica. - : Wiley. - 1600-0463. ; 112:11-12, s. 808-814
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Forskningsöversikt (refereegranskat)abstract
- Increased use of powerful PCR technology for the routine detection of pathogens has focused attention on the need for international validation and preparation of official non-commercial guidelines. Bacteria of epidemiological importance should be the prime focus, although a "validation infrastructure" once established could easily be adapted for PCR-based detection of viruses and parasites. The aim of standardization should be the widespread adoption of diagnostic PCR for routine pathogen testing. European experience provides the impetus for realization of this vision through preparation of quantitative reference DNA material and reagents, production of stringent protocols and tools for thermal cycler performance testing, uncomplicated sample preparation techniques, and extensive ring trials for assessment of the efficacy of selected matrix/pathogen detection protocols.
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| 2. |
- Lübeck, P S, et al.
(författare)
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Towards an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation.
- 2003
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Ingår i: Applied and Environmental Microbiology. - 0099-2240. ; 69:9, s. 5664-5669
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Tidskriftsartikel (refereegranskat)abstract
- As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.
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| 3. |
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| 4. |
- Rådström, Peter, et al.
(författare)
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Pre-PCR processing : Strategies to generate PCR-compatible samples
- 2004
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Ingår i: Molecular Biotechnology. - 1073-6085 .- 1559-0305. ; 26:2, s. 133-146
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Tidskriftsartikel (refereegranskat)abstract
- Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.
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| 5. |
- Rådström, Peter, et al.
(författare)
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Pre-PCR processing of samples.
- 2003
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Ingår i: Methods in Molecular Biology. - 1064-3745 .- 1940-6029. ; 216
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Tidskriftsartikel (refereegranskat)
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| 6. |
- Rådström, Peter, et al.
(författare)
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Pre-PCR processing strategies
- 2004
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Ingår i: PCR technology : current innovations. - 0849311845
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- A review describes the sample prepn. for biochem. methods, enrichment methods, immunol. methods, and phys. methods. The pretreatment of a complex biol. sample is crucial, and for successful PCR the following requirements have to be fulfilled: complete lack or low concn. of PCR-inhibitory components in the sample and sufficient concn. of target DNA. The aim of the pre-PCR treatment is to convert a complex biol. sample contg. the target microorganisms into PCR-amplifiable samples.
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| 7. |
- Wolffs, Petra, et al.
(författare)
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Impact of DNA polymerases and their buffer systems on quantitative real-time PCR
- 2004
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Ingår i: Journal of Clinical Microbiology. - 1098-660X. ; 42:1, s. 408-411
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Tidskriftsartikel (refereegranskat)abstract
- An investigation of the influence of five DNA polymerase-buffer systems on real-time PCR showed that the choice of both DNA polymerase and the buffer system affected the amplification efficiency as well as the detection window. The analytical repeatability of the data for different systems changed clearly, leading us to conclude that basing quantitative measurements on single-data-set standard curves can lead to significant errors.
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| 8. |
- Wolffs, Petra
(författare)
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Pre-PCR Processing Strategies for Quantitative Detection of Food-Borne Pathogens using Real-Time PCR
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Since the development of real-time PCR, the technology has been widely applied in the field of diagnostics. In comparison with conventional PCR it has opened up the possibility of accurate quantification of microorganisms in clinical, environmental and food samples. When employing real-time PCR and absolute quantification in biological samples, five important requirements should be fulfilled, namely (i) elimination of PCR inhibition, (ii) concentration of target nucleic acids or cells, (iii) conversion of heterogeneous samples into homogeneous PCR samples, (iv) avoidance of false-positive and false-negative results, and (v) enabling quantification. The objective of the work described in this thesis was to develop new pre-PCR processing strategies for quantification of pathogens in food, by altering the real-time PCR mixture and combining this with suitable sample treatment methods. The PCR mixture was modified by using alternative DNA polymerases and amplification facilitators to circumvent DNA inhibition and to obtain a good amplification efficiency. Furthermore, a new salt-insensitive probe system, based on a peptide nucleic acid thiazole orange conjugate, was studied. The resulting improvements in the PCR mixture were combined with a novel form of sample treatment called floatation which, in combination with quantitative (q) PCR, was used for absolute quantification of Yersinia enterocolitica and Campylobacter spp. in various food and clinical samples. Results showed that after floatation the sample matrix and the background flora could be separated from the target pathogen in such a way that PCR inhibition was minimized to levels comparable to that of purified DNA in Millipore water. Applying floatation to meat juice samples containing natural background flora and spiked with different concentrations of Y. enterocolitica, showed that absolute quantification of Y. enterocolitica was possible down to levels of 4.2 „e 103 CFU/ml even if an additional 1 ¡Ñ 106 CFU/ml BGF were added. Furthermore, the design of the floatation set-up ensured that false-positive results from free target DNA or dead cells were greatly reduced.
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| 9. |
- Wolffs, Petra, et al.
(författare)
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Quantification of Campylobacter spp. in chicken rinse samples by using flotation prior to real-time PCR
- 2005
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Ingår i: Applied and Environmental Microbiology. - 0099-2240. ; 71:10, s. 5759-5764
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Tidskriftsartikel (refereegranskat)abstract
- Real-time PCR is fast, sensitive, specific, and can deliver quantitative data; however, two disadvantages are that this technology is sensitive to inhibition by food and that it does not distinguish between DNA originating from viable, viable nonculturable (VNC), and dead cells. For this reason, real-time PCR has been combined with a novel discontinuous buoyant density gradient method, called flotation, in order to allow detection of only viable and VNC cells of thermotolerant campylobacters in chicken rinse samples. Studying the buoyant densities of different Campylobacter spp. showed that densities changed at different time points during growth; however, all varied between 1.065 and 1.109 g/ml. These data were then used to develop a flotation assay. Results showed that after flotation and real-time PCR, cell concentrations as low as 8.6 X 10(2) CFU/ml could be detected without culture enrichment and amounts as low as 2.6 X 10(3) CFU/ml could be quantified. Furthermore, subjecting viable cells and dead cells to flotation showed that viable cells were recovered after flotation treatment but that dead cells and/or their DNA was not detected. Also, when samples containing VNC cells mixed with dead cells were treated with flotation after storage at 4 or 20 degrees C for 21 days, a similar percentage resembling the VNC cell fraction was detected using real-time PCR and 5-cyano-2,3-ditolyl tetrazolium chloride-4',6'-diamidino-2-phenylindole staining (20% +/- 9% and 23% +/- 4%, respectively, at 4 degrees C; 11% +/- 4% and 10% +/- 2%, respectively, at 20 degrees C). This indicated that viable and VNC Campylobacter cells could be positively selected and quantified using the flotation method.
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| 10. |
- Wolffs, Petra, et al.
(författare)
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Rapid quantification of yersinia enterocolitica in pork samples by a novel sample preparation method, flotation, prior to real-time PCR
- 2004
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Ingår i: Journal of Clinical Microbiology. - 1098-660X. ; 42:3, s. 1042-1047
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Tidskriftsartikel (refereegranskat)abstract
- The development of real-time PCR thermal cycles in the late 1990s has opened up the possibility of accurate quantification of microorganisms in clinical, environmental, and food samples. However, a lack of suitable sample preparation methods that allow rapid quantification of the nucleic acids, remove PCR inhibitors, and prevent false-positive results due to DNA originating from dead cells has limited the use of quantitative PCR. We have used for the first time a new variant of density gradient centrifugation, called flotation, as a user-friendly sample preparation method prior to PCR. This paper describes the use of this sample preparation method, without DNA purification, for direct detection and quantification of Yersinia enterocolitica in PCR-inhibitory meat juice from pork. Flotation combined with qPCR could overcome PCR interference in juice from pork, as was shown by amplification efficiencies of 1.006 +/- 0.021 and 1.007 +/- 0.025, which are comparable to the amplification efficiency obtained for purified DNA samples (1.005 +/- 0.059). Applying flotation to meat juice samples containing natural background flora and spiked with different levels of Y. enterocolitica showed that direct quantification of Y enterocolitica was possible down to a level of at least 4.2 x 10(3) CFU per ml of meat juice, even in the presence of 10(6) CFU of background flora per ml. Finally, the results showed that samples containing large amounts of Y. enterocolitica DNA did not result in a positive PCR signal. This indicates that the risk of false-positive results due to detection of DNA originating from dead cells can be greatly reduced by using flotation prior to PCR.
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