| 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. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- 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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